aniridia ( an2 : omim 106210 ) is a panocular genetic disorder that is manifested by alterations in the structure and function of the eye , including a variable degree of iris hypoplasia and several other ocular features such as cataracts , glaucoma , corneal opacity , foveal hypoplasia and nystagmus . genetically , aniridia is the first human malformation to be recognized as stemming from an alteration in a paired box homeotic gene . the only known gene for aniridia , pax6 , was cloned from chromosome 11p13 , and , to date , no other genetic loci have been found to be associated with aniridia . in this study , we present the clinical and molecular genetic evaluations performed on two unrelated tunisian families with aniridia and congenital cataracts . two unrelated non - consanguineous tunisian families were referred to the department of congenital and hereditary disorders at charles nicolle hospital in tunis , tunisia , because multiple family members were affected with autosomal dominant aniridia associated with congenital cataracts ( figure 1 ) . the clinical diagnosis was confirmed by ophthalmologic examination , including visual acuity , slit - lamp examination , gonioscopy , optical coherence tomography and a history of cataract extraction ( if applicable ) . according to the classification of aniridia phenotypes , six of the seven affected patients were categorized as iris 6 ( complete aniridia ) . the seventh patient , ii8 , who belonged to family f2 , had a unilateral cataract of the right eye . two patients , ii8 ( from f1 ) and ii8 ( from f2 ) , had undergone cataract extraction . we noted that optical coherence tomography ( oct ) demonstrated the presence of foveal hypoplasia in the brother iii11 and the sister iii12 from family f1 , whose mother showed corneal dystrophy . all seven affected patients from the two families had normal muscle tone and normal signs on gonioscopy . however , most ophthalmologic features were not established for patient iii14 , who belonged to family f2 , because of poor cooperation . the study was performed in the laboratory of human genetics , faculty of medicine of tunis , tunisia , in accordance with the declaration of helsinki ; the ethics committee of charles nicolle hospital in tunis gave approval for this study . one hundred unrelated subjects , with no features of ocular anomalies , were recruited as controls . genomic dna of the affected and unaffected members from the two studied families was extracted from peripheral blood leukocytes using a standard proteinase - k protocol . all 14 exons and intron  exon junctions of the pax6 gene were amplified from genomic dna by polymerase chain reaction ( pcr ) using primers chosen by primer3 ( primer3-web / hldocs / inpat-040.htm ) . the amplification reactions were performed in an automated thermal cycle geneamp pcr system 9700 ( applied biosystems , foster city , ca , usa ) . the amplified products were purified ( zymo research dna clean & concentrator-25 kit , irvine , ca , usa ) and sequenced ( big dye terminator cycle sequencing ready reaction ; dna sequencing kit ; abi prism 3130 ) in the forward and reverse directions . sequencing results were visualized , and the data were analyzed using the sequencing analysis 5.2 and seqscape software ( applied biosystems ) . protein sequences among eight different species were aligned using the ucsc genome browser ( http://genome.ucsc.edu/ ) . the smart site ( simple modular architecture research tool : http://smart.embl-heidelberg.de/ ) was used to compare the sequence of a normal protein with a mutated one . by direct sequencing of the coding and flanking regions of pax6 , we identified a novel nonsense mutation ( c.265c > t ) in family f1 that was detected in exon 6 of the three affected members . the heterozygous mutation resulted in the substitution of a highly conserved glutamine codon for a termination codon ( p.q89x ) leading to a truncated protein . the c.265c > t mutation was not detected in the unaffected father ( ii7 ) or in any of the 100 control individuals from the same ethnic background , supporting the idea that this novel variation is a disease - causing mutation that segregates with the studied phenotype in family f1 . for the four affected individuals belonging to family f2 , we detected a heterozygous substitution ( c.718c > t ) in exon 9 of the pax6 gene leading to the replacement of an arginine codon for a termination codon ( p.r240x ) . this nonsense mutation , which generates a truncated protein , has previously been reported in patients with aniridia from different countries . the sequence alignment analysis for the novel nonsense mutation , c.265c > t , identified in family f1 showed that gln89 of the human pax6 protein ( homo sapiens , np_000271.1 ) was highly conserved in various species , including pan troglodytes , macaca mulatta , mus musculus , cavia porcellus , loxodonta africana , monodelphis domestica , gallus gallus and danio rerio . a smart analysis showed that the p.q89x mutation was located at the dna - binding paired domain of the pax6 protein , which generated a truncated protein with 88 amino acids and containing only a non - functional paired domain ( pd ) . most pax6 mutations reported so far lead to the truncation of encoded proteins . in the present study , the two identified pax6 mutations are nonsense mutations . the first one , c.265c > t , is a novel mutation detected in a tunisian family ( f1 ) with aniridia and congenital cataracts . this mutation leads to a truncated pax6 protein via the substitution of a highly conserved amino acid ( gln ) for a termination codon ( p.q89x ) . the p.q89x was not detected in 100 ethnically matched , healthy individuals and had not been previously reported according to the human pax6 mutation database ( http://www.hgu.mrc.ac.uk/softdata/pax6/ ) and lovd ( mrc human genetics unit lovd at mrc igmm , paired box gene 6 ) . this finding confirms that the sequence change is a disease - causing , co - segregating mutation that affects the dna - binding paired domain of the pax6 protein . this point mutation is an n - terminal mutation leading to a severe phenotype in the affected members of this family . consequently , the c.265c > t mutation affecting the pax6 s pd is inclined to cause pax6 haploinsufficiency through a loss - of - function mutation and produced complete congenital aniridia and congenital cataracts in the mother of family f1 and her two children . structurally , pax6 is composed of 422 amino acids , with 124 amino acids comprising the pd . the novel substitution of the glutamine codon for a stop codon at position 89 of the protein pax6 generates a truncated protein composed only of an aberrant pd of 88 amino acids . consequently , a haploinsufficiency of the pax6 protein is generated , in which the mutated pax6 protein does not have any transcriptional activity , and the remaining single normal copy of pax6 does not produce a functional threshold level of biologically active pax6 protein sufficient to initiate the transcription of its target genes . the father of family f2 presented with partial aniridia associated with a unilateral congenital cataract , while his three children were diagnosed with complete aniridia and bilateral congenital cataracts with other variable ocular anomalies . we identified the previously described c.718c > t ( p.r240x ) nonsense mutation in exon 9 of pax6 gene in this family . the p.r240x , which involves cpg dinucleotides , is among the most common nonsense mutation in pax6 
  and was reported to be pathogenic . it affects the homeodomain of the protein in that the premature insertion of a stop codon is predicted to result in nonsense - mediated decay and , consequently , a functionally null allele . previously , this point mutation was found in at least 31 patients worldwide , including the familial and sporadic cases reported in the human pax6 mutation database ; 26 of the patients have isolated aniridia , and the remaining 5 have associated ocular anomalies ( 4 with cataracts ) . the father , ii8 , from family f2 , presented with partial aniridia of the left eye ( < 180 ) and a congenital cataract of nuclear and cortical progressive type in the right eye . this phenotype could be explained by the wide expression of pax6 gene in the developing eye and its involvement in ocular morphogenesis , notably in lens development . this report confirms the phenomenon of variable phenotypes occurring within a family and indicates that factors other than the mutated pax6 protein may contribute to the complexity of anterior segment phenotypes . in summary , we identified a novel nonsense mutation ( p.q89x ) in a tunisian family with aniridia and congenital cataracts . additionally , we highlighted the predicted pathogenic effect of the reported nonsense mutation , p.r240x , in a second tunisian family with aniridia , congenital cataracts and variable ocular anomalies . these two mutations lead to truncated proteins and added to the large spectrum of nonsense mutations associated with aniridia .
the aim of this study was to identify the genetic defect that is responsible for aniridia and congenital cataracts in two tunisian families . 
 sequencing of the pax6 gene in family f1 detected a novel c.265c > t transition in exon 6 . 
 in family f2 , the previously described c.718c > t mutation in pax6 was detected in the four affected members . 
 this study adds new mutation to those previously reported in pax6 , providing further evidence for the genetic and phenotypic heterogeneity in individuals with aniridia ocular malformations .