Anophthalmia and microphthalmia (A/M) are developmental ocular malformations 
defined as the complete absence or reduction in size of the eye. A/M is a highly 
heterogeneous disorder with SOX2 and FOXE3 playing major roles in dominant and 
recessive pedigrees, respectively; however, the majority of cases lack a genetic 
etiology. We analyzed 28 probands affected with A/M spectrum (without mutations 
in SOX2/FOXE3) by whole-exome sequencing. Analysis of 83 known A/M factors 
identified pathogenic/likely pathogenic variants in PAX6, OTX2 and NDP in three 
patients. A novel heterozygous likely pathogenic variant in PAX6, c.767T>C, 
p.(Val256Ala), was identified in two brothers with bilateral microphthalmia, 
coloboma, primary aphakia, iris hypoplasia, sclerocornea and congenital 
glaucoma; the unaffected mother appears to be a mosaic carrier. While A/M has 
been reported as a rare feature, this is the first report of congenital primary 
aphakia in association with PAX6 and the identified allele represents the first 
variant in the PAX6 homeodomain to be associated with A/M. A novel pathogenic 
variant in OTX2, c.651delC, p.(Thr218Hisfs*76), in a patient with syndromic 
bilateral anophthalmia and a hemizygous pathogenic variant in NDP, c.293 C>T, 
p.(Pro98Leu), in two brothers with isolated bilateral microphthalmia and 
sclerocornea were also identified. Pathogenic/likely pathogenic variants were 
not discovered in the 25 remaining A/M cases. This study underscores the utility 
of whole-exome sequencing for identification of causative mutations in highly 
variable ocular phenotypes as well as the extreme genetic heterogeneity of A/M 
conditions.