Exome Sequencing Identification of a Novel Causal Mutation for Eyelid Dysplasia

Abstract

Purpose: Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is a complex eyelid malformation. A novel dominantly inherited BPES-like eyelid/conjunctival phenotype was seen in an Ashkenazi Jewish family. The causal gene for BPES -FOXL2- was sequenced, with no mutations detected. Deep sequencing was performed to identify a causal variant for this novel phenotype.

Methods: Six members were recruited for study (4 affected, 2 unaffected). The affected phenotype consisted of ptosis, lateral canthal dystopia, telecanthus, absent lower eyelid lashes, epicanthus tarsalis rather than inversus, normal horizontal palpebral fissure lengths, and atypical lateral conjunctival ciliated epibulbar keratinization. Pathology uniformly revealed inflamed conjunctiva. Whole exome sequencing was performed with 2 affected DNA samples. SNP and Variation Suite 7.5 software was used to filter single nucleotide variants (SNVs) and micro-insertions/deletions (indels). Public databases and internal exome controls were used to filter out known variants. PCR and sequencing primers were designed to confirm co-segregating variants with additional family members. Variant allelic frequencies were determined by genotyping 672 ethnically matched controls.

Results: An average 50X coverage depth for all coding regions was achieved. Eleven SNVs/indels passed filtering criteria. A segregating, novel heterozygous change (c.868A>G) was detected in GALNT2 (chromosome 1q42.13), causing an arginine to glycine (R290G) change in an evolutionarily conserved region. The variant was negative in 672 controls. cDNA expression panels validated GALNT2 presence in all human ocular and relevant systemic tissues. In silico prediction tools (XFold and SIFT/ANNOTATOR) of the p.R290G suggests both a moderate destabilizing effect of the crystal structure (2.47 kcal/mol), and affects protein function (score 0.01/0.00), respectively.

Conclusions: We identified a novel causal gene for a BPES-like eyelid/conjunctival dysplasia. GALNT2 functions in the mucin-type O-glycan biosynthesis pathway affecting mucin expression in conjunctival epithelial cells. GALNT2 is also involved in eyelid development through the epidermal growth factor receptor (EGFR). EGFR/MAPK/AP-1 signaling at the edge of eyelid keratinocytes is required for normal eyelid development. Whole exome sequencing is useful in causal gene discovery for rare ocular disorders.

Keywords: 526 eyelid • 474 conjunctiva • 539 genetics