Identification of <i>PITX3</i> mutations in individuals with various ocular developmental defects

<p><b>Background</b>: Congenital cataract displays large phenotypic (syndromic and isolated cataracts) and genetic heterogeneity. Mutations in several transcription factors involved in eye development, like <i>PITX3</i>, have been associated with congenital cataracts and anterior segment mesenchymal disorders.</p> <p><b>Materials and methods</b>: Targeted sequencing of 187 genes involved in ocular development was performed in 96 patients with mainly anophthalmia and microphthalmia. Additionally, Sanger sequencing analysis of <i>PITX3</i> was performed on a second cohort of 32 index cases with congenital cataract and Peters anomaly and/or sclereocornea.</p> <p><b>Results</b>: We described five families with four different <i>PITX3</i> mutations, two of which were novel. In Family 1, the heterozygous recurrent c.640_656dup (p.Gly220Profs*95) mutation cosegregated with eye anomalies ranging from congenital cataract to Peters anomaly. In Family 2, the novel c.669del [p.(Leu225Trpfs*84)] mutation cosegregated with dominantly inherited eye anomalies ranging from posterior embryotoxon to congenital cataract in heterozygous carriers and congenital sclereocornea and cataract in a patient homozygous for this mutation. In Family 3, we identified the recurrent heterozygous c.640_656dup (p.Gly220Profs*95) mutation segregating with congenital cataract. In Family 4, the <i>de novo</i> c.582del [p.(Ile194Metfs*115)] mutation was identified in a patient with congenital cataract, microphthalmia, developmental delay and autism. In Family 5, the c.38G>A (p.Ser13Asn) mutation segregated dominantly in a family with Peters anomaly, which is a novel phenotype associated with the c.38G>A variant compared with the previously reported isolated congenital cataract.</p> <p><b>Conclusions</b>: Our study unveils different phenotypes associated with known and novel mutations in <i>PITX3</i>, which will improve the genetic counselling of patients and their families.</p

Recurrent mutations in known autosomal recessive non-syndromic hearing loss (ARNSHL) genes in 6 Pakistani families.

<p>Acc. No., accession number of reference sequence; Chr, chromosome; Ex, exon; EVS, exome variant server; In, intron; SNPs, single nucleotide polymorphisms; Ref, references.</p

Pedigrees and the segregation of novel mutations in known deafness genes.

<p>Unfilled circles indicate unaffected females, unfilled squares indicate unaffected males, filled circles indicate affected females, filled squares indicate affected males, double lines represent consanguineous marriages, slashed line across the symbols indicate deceased individual, + indicates wild type allele, M indicates mutant allele.</p

Novel mutations identified in known genes for autosomal recessive non-syndromic hearing loss (ARNSHL) in the current study.

<p>Acc. No., accession number of reference sequence; Chr, chromosome; Ex, exon; EVS, exome variant server; hg19, human genome assembly 19; In, intron; NA, not applicable; ND, not determined; SNPs, single nucleotide polymorphisms; PhyloP, phylogenetic P-values; Polyphen, polymorphism phenotyping; SIFT, sorting intolerance from tolerance.</p

Spectrum of recurrent <i>GJB2</i> mutations in Pakistani families with autosomal recessive non-syndromic hearing loss (ARNSHL).

<p>As reference sequence NM_004004.5 was employed. EVS, exome variant server;</p>#<p>The pathogenicity of this mutation is controversial.</p

Predicted effect of mutation c.726C>G (p.Cys242Trp) on the three dimensional structure of TMPRSS3.

<p>A) Wild type protein structure with an intact disulphide bridge showing position of the mutated residue (magenta). B) Close-up view of the structure showing the wild type residue cysteine (green) and the mutant residue tryptophan (red). In case of the mutant residue there will be no disulphide bridge at this position.</p

Effect of <i>TMC1</i> intronic mutation c.362+18A>G using a minigene approach.

<p>Electropherogram of the partial cDNA sequence of RNA derived from cells transfected with the pCI-NEO with either the mutant or wildtype <i>TMC1</i> exon 8. The mutation leads to the insertion of 17bp at the 3′end of exon 8, which can be predicted to result in a premature stop codon in exon 9 (p.Glu122Tyrfs*10).</p

Effect of MYO15A c. 9948G>A using a minigene approach.

<p>An agarose gel containing RT-PCR products detected from HEK293T cells transfected with the wildtype and mutant minigene construct and a schematic representation of the identified splicing products. The RT-PCR products were verified by sequence analysis. The c.9948G>A mutation leads to skipping of exon 61.</p