Exome sequencing identifies genetic variants in anophthalmia and microphthalmia

Abstract

Anophthalmia and microphthalmia (A/M) are rare birth defects affecting up to 2 per 10,000 live births. These conditions are manifested by the absence of an eye or reduced eye volumes within the orbit leading to vision loss. Although clinical case series suggest a strong genetic component in A/M, few systematic investigations have been conducted on potential genetic contributions owing to low population prevalence. To overcome this challenge, we utilized DNA samples and data collected as part of the National Birth Defects Prevention Study (NBDPS). The NBDPS employed multi-center ascertainment of infants affected by A/M. We performed exome sequencing on 67 family trios and identified numerous genes affected by rare deleterious nonsense and missense variants in this cohort, including de novo variants. We identified 9 nonsense changes and 86 missense variants that are absent from the reference human population (Genome Aggregation Database), and we suggest that these are high priority candidate genes for A/M. We also performed literature curation, single cell transcriptome comparisons, and molecular pathway analysis on the candidate genes and performed protein structure modeling to determine the potential pathogenic variant consequences on PAX6 in this disease.

Keywords: congenital abnormalities; genetic epidemiology; newborn eye abnormalities.

Figure 1.. Analysis schematic.

Overview of the approach to identifying candidate genes from 67 probands. Following GATK Best Practice, we applied the variant quality score recalibration (VQSR) procedure to refine the raw variants from the initial genotype calls. To define rare variants, we considered those not seen in the Genome Aggregation Database (gnomAD).

Figure 2.. Single-cell transcriptome data analysis of candidate genes across retina developmental stages.

a. In the first trimester, the identified eight genes displayed significantly increased expression relative to the background control genes in AC/HC_Precursors (N= 695), Amacrine Cells (N= 253), BC/Photo_Precursors, Cones (N= 689), Horizontal Cells (N= 1,883), Neurogenic Cells (N= 1,123) and Retinal Ganglion Cells (N= 6,297). The p values were calculated by Wilcoxon rank-sum test, followed by Benjamini-Hochberg correction. Cell types in red font along the x-axis had a false discovery rate (FDR) <0.05. Abbreviations: AC/HC, amacrine cell/horizontal cell; BC/Photo, bipolar cell/photoreceptor. b. In the second trimester, the identified eight genes displayed significantly increased expression relative to the background control genes in AC/HC_Precursors (N= 1,039), Amacrine Cells (N= 10,706), BC/Photo_Precursors (N= 1,825), Bipolar Cells (N= 5,949), Cones (N= 3,985), Horizontal Cells (N= 4,618), Neurogenic Cells (N= 2,197), Retinal Ganglion Cells (N= 2,662) and Muller Glia (N= 343). The p values were calculated by Wilcoxon rank-sum test, followed by Benjamini-Hochberg correction. Cell types in red font along the x-axis had a false discovery rate (FDR) <0.05. a. & b. The bottom and top of the boxes denote the first and third quartiles, respectively. The whiskers indicate the minimal value within 1.5 interquartile range (IQR) of the lower quartile and the maximum value within 1.5 IQR of the upper quartile. The plus symbols represent outliers. The black dashed lines indicate the lower and upper limits of the regions with regular scale. Outliers outside of the black dashed lines are visualized with compressed scale in the regions surrounded by gray lines for better visualization. c. CHD7 is ubiquitously expressed across all retinal cell types, despite a stronger pattern in retinal progenitor cells, cones and BC/photo precursors. Red and grey colors represent high and low read number in CHD7 expression, respectively. Uniform manifold approximation and projection (UMAP) is an efficient dimension reduction algorithm commonly used in single-cell RNA sequencing analysis. d. XKR4 displayed strong specificity in the developing retina only in very few cell types, including retinal ganglion cells, amacrine cells, horizontal cells, and cones. Red and grey colors represent high and low XKR4 expression, respectively.

Figure 3.. Protein structure analysis of a potential deleterious missense variant in PAX6.

a. The gene structure of transcription factor PAX6. The potential pathogenic variant (p.G19E) altered the wildtype amino acid from a glycine (G) residue into a glutamic acid (E) in the paired DNA binding domain of PAX6. PST represents a carboxyl-terminal transactivation domain rich of proline (P), serine (S), and threonine (T). b. The p.G19E potential pathogenic variant of PAX6 had a significant effect in loss of molecular flexibility in the N-terminus. 3D structure of mutated PAX6, blue and white colors stand for high and low rigidification. The zoomed in region indicates the differences between wild type and mutant in mutated residue location, red represents hydrogen bonds and light-green represents residues. The table shows the prediction outcomes of interatomic interactions.