A novel mutation in the OAR domain of PITX3 associated with congenital posterior subcapsular cataract

Three members of a four-generation Chinese family were enrolled in this study, including the proband (IV:3) and her parents (the father III:5 and the mother III:6). Two of them were affected with autosomal dominant congenital cataract (the proband IV:3 and her father III:5). They received a comprehensive ophthalmological examination at the Department of Ophthalmology of the Eye & ENT Hospital of Fudan University. This examination included tests of uncorrected visual acuity (UCVA) and best corrected visual acuity (BCVA), Goldmann applanation tonometry, and detailed slit-lamp and fundus examinations. Others family members were not enrolled, but their affected status was obtained from the medical records of the local hospital from previous ophthalmological examinations.

Five milliliters of peripheral venous blood from the three participants was collected in tubes containing EDTA. DNA was isolated from the whole-blood samples using a Gentra Puregene Blood Kit (Qiagen, Valencia, CA) in accordance with the manufacturer’s protocol and was stored at − 20 °C until used for sequencing. No DNA from the other family members was available for targeted mutation testing.

All three DNA samples were subjected to analysis using panel-based NGS that involved a targeted gene region capture protocol to identify genetic defects. This protocol and the capture probes were custom designed and produced by Beijing Genomics Institute (BGI, Shenzhen, China), as previously reported. [11] The output sequence data were aligned to the reference human genome UCSC hg38 using Burrows-Wheeler Aligner, version 0.7.10. The variants were filtered to eliminate benign variants with MAF (minor allele frequency) > 0.1% in the 1000 Genomes dataset, and the dbSNP, EXAC, Tumor Heart Freq_Hom, ESP6500, and internal databases. All 790 genes were considered equally. Finally, variant prioritization was performed by combining the total depth, quality score, MAF, potential deleterious effect and the existence of mutation reports in common databases, such as The Human Gene Mutation Database (HGMD), The Retinal Information Network (RetNet), ClinVar, and Online Mendelian Inheritance in Man (OMIM). Variants were classified as benign, likely benign, pathogenic, likely pathogenic or novel variants of uncertain clinical significance according to the American College of Medical Genetics and Genomics (ACMG) guidelines [12].

The pedigree of the family enrolled in this study is shown in Fig. 1 (Only the proband and her parents were enrolled in the study). There are four generations that include 11 affected individuals, five unaffected individuals and 8 spouses. The proband (IV:3) was born with bilateral congenital cataract. Her BCVA was 0.4 (logMar unit) in her right eye and 1.0 (logMar unit) in her left eye when she was first referred to our hospital at the age of 10. Posterior subcapsular lenticular opacity was observed in both eyes, with heavier opacity in the left eye than in the right eye (Fig. 2 a, b, c and d). Cataract extraction with posterior capsulotomy, anterior vitrectomy and intraocular lens (IOL) implantation was performed. One year after surgery, both eyes reached a BCVA of 0.1 (logMar unit), with a clear optical axis. The proband’s father (III:5) had also suffered from congenital cataract since childhood. He underwent cataract extraction with IOL implantation at the age of 18, resulting in a bilateral BCVA of 0.7 (logMar unit). He presented with pseudophakia in both eyes and mild IOL dislocation in the right eye (Fig. 2 E and F). The patients had no ocular or systemic abnormalities other than cataract.

After the data acquisition and analysis, a total of 14,076 raw variants were found in the 790 genes, and 211 variants (Additional file 1: Table S1) met the filtering criteria. Based on valuable information in published literatures, we included the genes that were of autosomal dominant genetic inheritance pattern and congenital cataract. Finally, one deletion mutation (c.797_814del, p.Ser266_Ala271del) in PITX3 was identified as a potentially pathogenic mutation. This mutation was identified in IV:3 and III:5 (Fig. 3) and was validated by Sanger sequencing. The PITX3 deletion mutation had not been recorded in the public mutation GnomAD database (http://​gnomad.​broadinstitute.​org/​). The mutation is classified as an uncertain clinical significance mutation according to the ACMG guidelines (criteria PM2, PM4, and PP1). It is located within the otp/aristaless/rax (OAR) domain at the C-terminus of the protein (Fig. 4), which functions in DNA binding mediated by protein-protein interactions. This mutation would result in a deletion of 6 amino acid residues at the C-terminus of the protein (266–271, from a total of 302).