strabismus , also referred to as squint , presents as a misalignment of the eyes . strabismus is also a feature of several syndromes , such as congenital fibrosis of extraocular muscles , duane retraction syndrome , and chronic progressive external ophthalmoplegia . population - based studies show that the familial clustering of strabismus has been observed , and the incidence of specific types of strabismus differs , in different racial groups , which suggests that the etiology of strabismus has genetic factors [ 38 ] . michaelides et al . studied the genetics of strabismus and found that it was related to mitochondrial cytopathies and cranial nerve misrouting , which indicated that strabismus has a significant genetic component . however , significant genetic heterogeneity makes it difficult to determine the mode of inheritance and the relevant causative genes . in a large family with nonsyndromic strabismus , parikh et al . found a presumptive strabismus susceptibility locus to chromosome 7p22.1 with a multipoint lod score of 4.51 under a model of recessive inheritance . several studies have identified the causative gene mutations associated with strabismus - related disorders , such as congenital stationary night blindness , intellectual disability , and kaufman oculocerebrofacial syndrome . however , the identity of the strabismus susceptibility genes has been enigmatic and the genetic mechanisms involved in the pathogenesis of strabismus remain poorly understood . mutation screening of candidate genes may be necessary to localize genes that predispose humans to strabismus . exome sequencing is a powerful and cost - effective tool for dissecting the genetic basis of diseases [ 1618 ] . in the present study , we investigated a chinese strabismus pedigree with the parents unaffected and 2 offspring affected . by whole - exome sequencing , we identified 2 mutations c.a3257 g ( p.e1086 g ) in ahi1 and c.a914 g in neb as the most likely causative mutations of the disorder . of interest , biallelic pathogenic variant in ahi1 gene can cause joubert syndrome - related disorders with oculomotor apraxia characteristics . eye examinations focused on the measurement of visual acuity ( unaided and best - corrected ) and refractive status . his unaided visual acuity was 1.0 in the right eye and 1.0 in the left eye . the proband s sister was also diagnosed with comitant exotropia ( ii2 ) ( figure 1a ) . moreover , neither of them had any other symptoms such as nystagmus or joubert - like symptoms or any sign of hypotonia or vision / retinal . additionally , their parents , grandparents , and maternal grandparents were unaffected . based on these findings , we considered that the etiology of strabismus in this family had a genetic factor . therefore , the proband and his parents were enrolled for exome sequencing screening ( figure 1a ) . for the purpose of biological investigations , the study was approved by the ethics committee of the affiliated hospital of qingdao university ( 2015 - 012 ) and complied with the declaration of helsinki principles . venous blood was drawn from the affected individual ( ii-1 ) and 2 unaffected individuals ( i1 and i2 ) within the examined strabismus pedigree , then stored in tubes containing edta . all dna samples were prepared from the venous blood of the available patients by standard procedures . the qualified genomic dna was used to perform exome target enrichment using the agilent sureselect human all exon 50 mb exon kit ( agilent technologies , santa clara , ca , usa ) . the captured whole exomes were sequenced on the platform of illumina hiseq 2500 sequencer ( illumina , san diego , ca , usa ) . for each sample , sequencing reads with paired - end 125-bp long and mean coverage of 100x was generated . after filtering the adapter , contaminating reads and low - quality reads , the clean paired reads were then mapped to reference human genome sequence ( hg19 ) using the burrows - wheeler alignment tool ( bwa ) , generating the sequence alignment / map ( sam ) file . pcr duplicate reads were further marked and removed using the picard software program ( version 1.07 ) . genome analysis toolkit ( gatk )   and mutect software   were used to detect the single - nucleotide variants ( snvs ) , insertions , and deletions within genome - wide regions . to obtain the important candidate genes , the program annovar ( http://www.openbioinformatics.org/annovar/ ) was used to annotate the variants of the information from various genetic variation databases . based on the reported variant frequencies , the common variants were first excluded with the minor allele frequency ( maf ) greater than 0.01 represented in the 1000 genomes project . according to the variant location within genes , the variants in the coding region were given higher priority , and the variants that altered the coding sequence ( nonsynonymous ) were selected . the deleteriousness of selected variants was subsequently predicted by various bioinformatics programs ( sift , polyphen2 , lrt , mutationtaster , mutationassessor , fathmm , and radialsvm , lr ) , and the variants were retained whose changes to the protein were damaging . according to the autosomal recessive inheritance pattern , only the variants which were homozygous in the affected individual and heterozygous in the unaffected members were screened . moreover , we required at least 5x sequence coverage at any given position for genotype calling . genomic dna was prepared from peripheral blood samples of the original 3 individuals who underwent exome sequencing and 5 additional sporadically affected individuals . primer - premier 5.0 ( premier biosoft international , palo alto , ca , usa ) was used to design oligonucleotide primer sets for the variants from whole - exome sequencing . pcr reactions and sanger sequencing were performed by majorbio company ( shanghai , china ) . conservation of affected residue was analyzed using the ucsc genome browser ( http://genome-asia.ucsc.edu/index.html ) and visualized using mega7.0 . the protein conserved domains were further identified using the conserved domain search service ( http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi ) . eye examinations focused on the measurement of visual acuity ( unaided and best - corrected ) and refractive status . his unaided visual acuity was 1.0 in the right eye and 1.0 in the left eye . the proband s sister was also diagnosed with comitant exotropia ( ii2 ) ( figure 1a ) . moreover , neither of them had any other symptoms such as nystagmus or joubert - like symptoms or any sign of hypotonia or vision / retinal . additionally , their parents , grandparents , and maternal grandparents were unaffected . based on these findings , we considered that the etiology of strabismus in this family had a genetic factor . therefore , the proband and his parents were enrolled for exome sequencing screening ( figure 1a ) . for the purpose of biological investigations , the study was approved by the ethics committee of the affiliated hospital of qingdao university ( 2015 - 012 ) and complied with the declaration of helsinki principles . venous blood was drawn from the affected individual ( ii-1 ) and 2 unaffected individuals ( i1 and i2 ) within the examined strabismus pedigree , then stored in tubes containing edta . all dna samples were prepared from the venous blood of the available patients by standard procedures . the qualified genomic dna was used to perform exome target enrichment using the agilent sureselect human all exon 50 mb exon kit ( agilent technologies , santa clara , ca , usa ) . the captured whole exomes were sequenced on the platform of illumina hiseq 2500 sequencer ( illumina , san diego , ca , usa ) . for each sample , sequencing reads with paired - end 125-bp long and mean coverage of 100x was generated . after filtering the adapter , contaminating reads and low - quality reads , the clean paired reads were then mapped to reference human genome sequence ( hg19 ) using the burrows - wheeler alignment tool ( bwa ) , generating the sequence alignment / map ( sam ) file . pcr duplicate reads were further marked and removed using the picard software program ( version 1.07 ) . genome analysis toolkit ( gatk )   and mutect software   were used to detect the single - nucleotide variants ( snvs ) , insertions , and deletions within genome - wide regions . to obtain the important candidate genes , the resulting variants were annotated and filtered systematically . the program annovar ( http://www.openbioinformatics.org/annovar/ ) was used to annotate the variants of the information from various genetic variation databases . based on the reported variant frequencies , the common variants were first excluded with the minor allele frequency ( maf ) greater than 0.01 represented in the 1000 genomes project . according to the variant location within genes , the variants in the coding region were given higher priority , and the variants that altered the coding sequence ( nonsynonymous ) were selected . the deleteriousness of selected variants was subsequently predicted by various bioinformatics programs ( sift , polyphen2 , lrt , mutationtaster , mutationassessor , fathmm , and radialsvm , lr ) , and the variants were retained whose changes to the protein were damaging . according to the autosomal recessive inheritance pattern , only the variants which were homozygous in the affected individual and heterozygous in the unaffected members were screened . moreover , we required at least 5x sequence coverage at any given position for genotype calling . genomic dna was prepared from peripheral blood samples of the original 3 individuals who underwent exome sequencing and 5 additional sporadically affected individuals . primer - premier 5.0 ( premier biosoft international , palo alto , ca , usa ) was used to design oligonucleotide primer sets for the variants from whole - exome sequencing . pcr reactions and sanger sequencing were performed by majorbio company ( shanghai , china ) . conservation of affected residue was analyzed using the ucsc genome browser ( http://genome-asia.ucsc.edu/index.html ) and visualized using mega7.0 . the protein conserved domains were further identified using the conserved domain search service ( http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi ) . genomic dna from i : 1 , i : 2 and ii : 2 were analyzed by whole - exome sequencing ( wes ) . for each sample , an average of 9.67 gb raw data were generated with paired - end 125-bp length . above 99% clean reads were then aligned to the human reference sequence ( hg19 ) ( table 1 ) . a total of 567 705 single - nucleotide variations ( snvs ) were identified by variant detection with the genome analysis toolkit ( gatk ) and mutect software . among the variants identified by wes , we first removed variants with minor allele frequency > 0.01 in the 1000 genomes project , resulting in 118 542 variants , and 3191 variants were located in the exonic region . further filtering of synonymous variants led to 1851 loss - of - function variants , which altered the coding sequence and were more likely associated with the disease . based on the recessive - inherited mode of the strabismus pedigree , only 2 snvs fulfilled our filtering criteria : c. 914a - g mutation in neb ( chr2 : 151485334 .. 151734487 ) and c. 3257a - g mutation in ahi1 ( chr6 : 135283972 .. 135497775 ) . the proband of ii : 1 was homozygous for these mutations , and his clinically unaffected parents were heterozygous for these variants . variant frequencies were 0.0228 for mutation in neb and 0.0357 for mutation in ahi1 in the population of east asian ancestry from the 1000 genomes project . the c.3257a - g mutation in ahi1 resulted in p.1086e - g change , which was predicted to be damaging by polyphen2 . we searched the literature and found that biallelic pathogenic variants in ahi1 gene were identified in individuals with joubert syndrome - related disorders , which are characterized by cerebellar ataxia , oculomotor apraxia , hypotonia , neonatal breathing abnormalities , and psychomotor delay . it is reported that the expression of neb is remarkably decreased in strabismic extraocular muscles compared to normal muscles . therefore , we considered c.3257a - g mutation in ahi1 and c.914a - g mutation in neb as the most likely causal variants in this strabismus pedigree . to further confirm the variants of c.3257a - g mutation in ahi1 in strabismus , sanger sequencing was performed in 10 individuals with the basic type of strabismus and in 3 healthy individuals . the results showed that the mutation was not observed in other individuals with strabismus , indicating the genetic heterogeneity of strabismus . however , the mutation was found to be a homozygous mutation in his sister , which was consistent with the proband . the protein in 1086 locus of ahi1 protein sequence is highly conserved across species ( human , rhesus monkey , mouse , dog , and elephant ) . however , we found the rare mutation of c.3257a - g in ahi1 ( p.e1086 g ) ( figure 1b ) in this study . searching for conserved domains within the ahi1 protein indicated that the altered amino acid residue ( p.e1086 g ) was located in the conserved src homology 3 ( sh3 ) domain of ahi1 ( figure 1c ) . sh3 domains are protein interaction domains that bind to proline - rich ligands with moderate affinity and selectivity , and bind preferentially to pxxp motifs . they play versatile and diverse roles in the cell , including the regulation of enzymes , changing the subcellular localization of signaling pathway components , and mediating the formation of a multiprotein complex assembly . genomic dna from i : 1 , i : 2 and ii : 2 were analyzed by whole - exome sequencing ( wes ) . for each sample , an average of 9.67 gb raw data were generated with paired - end 125-bp length . above 99% clean reads were then aligned to the human reference sequence ( hg19 ) ( table 1 ) . a total of 567 705 single - nucleotide variations ( snvs ) were identified by variant detection with the genome analysis toolkit ( gatk ) and mutect software . among the variants identified by wes , we first removed variants with minor allele frequency > 0.01 in the 1000 genomes project , resulting in 118 542 variants , and 3191 variants were located in the exonic region . further filtering of synonymous variants led to 1851 loss - of - function variants , which altered the coding sequence and were more likely associated with the disease . based on the recessive - inherited mode of the strabismus pedigree , only 2 snvs fulfilled our filtering criteria : c. 914a - g mutation in neb ( chr2 : 151485334 .. 151734487 ) and c. 3257a - g mutation in ahi1 ( chr6 : 135283972 .. 135497775 ) . the proband of ii : 1 was homozygous for these mutations , and his clinically unaffected parents were heterozygous for these variants . variant frequencies were 0.0228 for mutation in neb and 0.0357 for mutation in ahi1 in the population of east asian ancestry from the 1000 genomes project . the c.3257a - g mutation in ahi1 resulted in p.1086e - g change , which was predicted to be damaging by polyphen2 . we searched the literature and found that biallelic pathogenic variants in ahi1 gene were identified in individuals with joubert syndrome - related disorders , which are characterized by cerebellar ataxia , oculomotor apraxia , hypotonia , neonatal breathing abnormalities , and psychomotor delay . it is reported that the expression of neb is remarkably decreased in strabismic extraocular muscles compared to normal muscles . therefore , we considered c.3257a - g mutation in ahi1 and c.914a - g mutation in neb as the most likely causal variants in this strabismus pedigree . to further confirm the variants of c.3257a - g mutation in ahi1 in strabismus , sanger sequencing was performed in 10 individuals with the basic type of strabismus and in 3 healthy individuals . the results showed that the mutation was not observed in other individuals with strabismus , indicating the genetic heterogeneity of strabismus . however , the mutation was found to be a homozygous mutation in his sister , which was consistent with the proband . the protein in 1086 locus of ahi1 protein sequence is highly conserved across species ( human , rhesus monkey , mouse , dog , and elephant ) . however , we found the rare mutation of c.3257a - g in ahi1 ( p.e1086 g ) ( figure 1b ) in this study . searching for conserved domains within the ahi1 protein indicated that the altered amino acid residue ( p.e1086 g ) was located in the conserved src homology 3 ( sh3 ) domain of ahi1 ( figure 1c ) . sh3 domains are protein interaction domains that bind to proline - rich ligands with moderate affinity and selectivity , and bind preferentially to pxxp motifs . they play versatile and diverse roles in the cell , including the regulation of enzymes , changing the subcellular localization of signaling pathway components , and mediating the formation of a multiprotein complex assembly . strabismus is characterized as a misalignment of the eyes , which is also a feature of several syndromes such as congenital fibrosis of extraocular muscles . family studies suggest that there is a strong genetic component to the etiology of strabismus [ 1,68 ] the advent of whole - exome sequencing technologies is a well - justified strategy for discovering candidate causative genes underlying genetic disease phenotypes . in this study , we aimed our efforts in this direction , and whole - exome sequencing was performed on the affected individual and his parents in a chinese strabismus pedigree , identifying 2 mutations in the genes of ahi1 and neb , which may be associated with strabismus . additionally , we detected the ahi1 homozygous mutation in the affected individual in this study . the inheritance patterns of strabismus are complex and are associated with complex sensory and motor pathways in the retina , thalamus , visual cortex , and brainstem [ 2527 ] . herein , genetic analysis was conducted on a chinese strabismus pedigree , and a mutation in ahi1 was identified . the structure of the ahi1 gene contains at least 33 exons and spans 213.7 kb . genomewide screening of 5 pedigrees with autosomal recessive joubert syndrome-3 identified 3 independent mutations in the ahi1 gene . using next - generation sequencing , najmabadi also identified homozygosity for a nonsense and a missense mutation in the ahi1 gene in affected members of 2 families with joubert syndrome-3 . pathogenic variants in ahi1 were identified in 7.3% of 137 persons with joubert syndrome . moreover , about 80% of them had retinal dystrophy . ahi1 expression is required for the development of cerebellar and cortical structure , and its missense and frameshift mutations were identified in families with joubert syndrome plus cortical polymicrogyria . additionally , joubert syndrome has been reported to be genetically heterogeneous with mutations in ahi1 , suggesting that this genotypic variant of joubert syndrome may be related to functional abnormalities of the cerebellum and brain stem . the ahi1 protein includes a sh3 domain , a coiled - coil domain , and 6 wd40 repeats . the ahi1 mutations occurred in a phenotype - specific group of joubert plus retinal abnormalities , and most of the mutations abolished all of the sh3 domain . consistent with that , we also found that the altered amino acid residue ( p.e1086 g ) was located in the conserved sh3 domain of ahi1 , suggesting its potential roles in strabismus . previous studies reported that approximately 1520% of strabismus was associated with global central nervous system ( cns ) defects rather than with ocular disorders . it has been suggested that the ipsilateral non - decussating projection from the temporal retina is suppressed in strabismus , indicating that the cns and retina play an important role in the pathology of strabismus . functional studies on mice showed that ahi1-null mice had defects of retinal outer segment morphogenesis and mislocalization of opsin in photoreceptors , which contributed to the loss of photoreceptors . the protein encoded by ahi1 ( also termed jouberin ) is involved in canonical wnt signaling . lancaster found that ahi1-mutant mice showed cerebellar hypoplasia and the wnt reporter activity in the developing cerebellum was decreased . these findings suggest that our variant in ahi1 may contribute to strabismus phenotype in this chinese pedigree . in this study neb encodes nebulin , which accounts for 3% to 4% of the total myofibrilla protein and regulates the length of actin filaments and contraction strength [ 4446 ] . it has been reported that neb has significantly down - regulated expression in strabismic extraocular muscles compared to normal muscles , which further demonstrates the relationship between neb and strabismus . our results defined 2 relevant genes , ahi1 and neb , that may predispose to strabismus , possibly through an additive effect . our finding revealed the high degree of genetic heterogeneity inherent in the strabismus phenotype , as demonstrated by parikh et al . , who discovered that the presumptive strabismus susceptibility locus to chromosome 7p22.1 was not presented in 6 other multiplex families . we conclude that this chinese strabismus pedigree best fits a model of autosomal recessive disease transmission .