Genetic analysis using next-generation sequencing and multiplex ligation probe amplification in Chinese aniridia patients

Abstract

Background: Congenital aniridia is a rare pan-ocular disease characterized by complete irideremia, partial iridocoloboma. The progressive nature of aniridia is frequently accompanied by secondary ocular complications such as glaucoma and aniridia-associated keratopathy, which can lead to severely impaired vision or blindness. The genetic basis of aniridia has been the subject of numerous studies, leading to the development of innovative therapeutic options based on PAX6 nonsense mutations. Specific knowledge of the genetics of aniridia has become increasingly important. To report the clinical features, elucidate the genetic etiology, and reveal the mutational spectrum of congenital aniridia in the Chinese population, sixty patients with congenital aniridia from 51 families were recruited. Candidate genes associated with developmental eye diseases were identified and analyzed using panel-based next-generation sequencing (NGS), and mutations were confirmed through polymerase chain reaction and Sanger sequencing. Multiplex ligation probe amplification (MLPA) of PAX6 and FOXC1 was performed to detect copy number variations in the patients without intragenic mutations.

Results: Clinical examination revealed complete iris hypoplasia in 58 patients and partial iris hypoplasia in two patients. Additionally, two patients were diagnosed with Wilms' tumor-aniridia-genital anomalies-retardation syndrome and nephroblastoma. By combining panel-based NGS and MLPA, 43 intragenic mutations or deletions of PAX6, FOXC1, and BCOR were identified in 59 patients, including 33 point mutations (76.7%) in 43 patients and 10 deletions (23.3%) in 16 patients. The total detection rate was 98.3%. Phenotypic variation was observed between and within families.

Conclusions: Variations in PAX6 and its adjacent regions were the predominant causes of aniridia in China. In addition to intragenic point mutations in PAX6, deletion of PAX6 or its adjacent genes is a common cause of congenital aniridia. Furthermore, FOXC1 is an important gene associated with congenital aniridia. The combination of panel-based NGS and MLPA significantly enhanced the detection rate of gene mutations in patients with congenital aniridia.

Keywords: BCOR; FOXC1; PAX6; Congenital aniridia; Multiplex ligation probe amplification; Panel-based next-generation sequencing.

Fig. 1

Typical anterior segment photographs of patients with congenital aniridia. A and B demonstrate complete hypoplasia of the iris in patients No. 4 (A) and No. 22 (B). C and D show partial iris hypoplasia or iridocoloboma with residual circular peripheral iris in patients No. 4 − 2 (C) and No. 30 (D). E and F show characteristic lenticular opacities in patients No. 19 (E) and No. 20 (F)

Fig. 2

Images of glaucoma, foveal hypoplasia, and congenital aniridia—anterior segment, fundus, OCT, UBM. (A) cornea edema and opacity in patient No. 36. (B) large cup-disc ratio and pale optic nerve in patient No. 27 − 2. (C) no macular reflex halo in patient No. 21. (D) flat macular fovea in patient No. 30 − 2. (E) only iris stump and unclear scleral process in patient No. 44

Fig. 3

Novel PAX6 mutations identified in this study. A-K: sequencing chromatograms of c.49_61del, c.1154delC, c.356delG, c.102_103del, c.65_66insTGCC, c.683–2 A > G, c.357 + 2insATAACA, c.808 A > T, c.140 A > T, c.781dupC and c.531 C > A in PAX6 gene

Fig. 4

The mutation c.4262G > A in BCOR gene identified in this study. A-C: sequencing chromatograms of No. 44 (proband), No. 44 − 2 (father), and No. 44 − 3 (mother), respectively

Fig. 5

Representative MLPA figures of PAX6 and adjacent genes. The blue columns with red top dots represent the deleted probes with peak areas below 0.65. The blue columns within grey areas indicate the control probes

Fig. 6

MLPA figures of FOXC1 deletions. The blue columns with red top dots depict the deleted probes with peak areas below 0.65. The blue columns within grey areas represent the control probes