Next-generation sequencing strategies have resulted in mutation detection rates of 21% to 61% in small cohorts of patients with microphthalmia, anophthalmia and coloboma (MAC), but despite progress in identifying novel causative genes, many patients remain without a genetic diagnosis. We studied a cohort of 19 patients with MAC who were ascertained from a population with high rates of consanguinity. Using single nucleotide polymorphism (SNP) arrays and whole exome sequencing (WES), we identified one pathogenic variant in TENM3 in a patient with cataracts in addition to MAC. We also detected novel variants of unknown significance in genes that have previously been associated with MAC, including KIF26B, MICU1 and CDON, and identified variants in candidate genes for MAC from the Wnt signaling pathway, comprising LRP6, WNT2B and IQGAP1, but our findings do not prove causality. Plausible variants were not found for many of the cases, indicating that our current understanding of the pathogenesis of MAC, a highly heterogeneous group of ocular defects, remains incomplete.

The Integrator complex subunit 1 (INTS1) is a component of the integrator complex that comprises 14 subunits and associates with RPB1 to catalyze endonucleolytic cleavage of nascent snRNAs and assist RNA polymerase II in promoter-proximal pause-release on protein-coding genes. We present five patients, including two sib pairs, with biallelic sequence variants in INTS1. The patients manifested absent or severely limited speech, an abnormal gait, hypotonia and cataracts. Exome sequencing revealed biallelic variants in INTS1 in all patients. One sib pair demonstrated a missense variant, p.(Arg77Cys), and a frameshift variant, p.(Arg1800Profs*20), another sib pair had a homozygous missense variant, p.(Pro1874Leu), and the fifth patient had a frameshift variant, p.(Leu1764Cysfs*16) and a missense variant, p.(Leu2164Pro). We also report additional clinical data on three previously described individuals with a homozygous, loss of function variant, p.(Ser1784*) in INTS1 that shared cognitive delays, cataracts and dysmorphic features with these patients. Several of the variants affected the protein C-terminus and preliminary modeling showed that the p.(Pro1874Leu) and p.(Leu2164Pro) variants may interfere with INTS1 helix folding. In view of the cataracts observed, we performed in-situ hybridization and demonstrated expression of ints1 in the zebrafish eye. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 to make larvae with biallelic insertion/deletion (indel) variants in ints1. The mutant larvae developed typically through gastrulation, but sections of the eye showed abnormal lens development. The distinctive phenotype associated with biallelic variants in INTS1 points to dysfunction of the integrator complex as a mechanism for intellectual disability, eye defects and craniofacial anomalies.

The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.