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Autosomal dominant North Carolina macular dystrophy (NCMD) is believed to represent a failure of macular development. The disorder has been linked to two loci, MCDR1 (chromosome 6q16) and MCDR3 (chromosome 5p15-p13). Recently, non-coding variants upstream of PRDM13 (MCDR1) and a duplication including IRX1 (MCDR3) have been identified. However, the underlying disease-causing mechanism remains uncertain. Through a combination of sequencing studies on eighteen NCMD families, we report two novel overlapping duplications at the MCDR3 locus, in a gene desert downstream of IRX1 and upstream of ADAMTS16. One duplication of 43 kb was identified in nine families (with evidence for a shared ancestral haplotype), and another one of 45 kb was found in a single family. Three families carry the previously reported V2 variant (MCDR1), while five remain unsolved. The MCDR3 locus is thus refined to a shared region of 39 kb that contains DNAse hypersensitive sites active at a restricted time window during retinal development. Publicly available data confirmed expression of IRX1 and ADAMTS16 in human fetal retina, with IRX1 preferentially expressed in fetal macula. These findings represent a major advance in our understanding of the molecular genetics of NCMD and provide insights into the genetic pathways involved in human macular development.

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs∗63), c.1996C>T (p.Arg666∗), c.2632G>T (p.Glu878∗), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies.

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.

Exome sequence analysis of affected individuals from two families with autosomal-dominant inheritance of coloboma identified two different cosegregating heterozygous nonsense mutations (c.370C>T [p.Arg124*] and c. 1066G>T [p.Glu356*]) in YAP1. The phenotypes of the affected families differed in that one included no extraocular features and the other manifested with highly variable multisystem involvement, including hearing loss, intellectual disability, hematuria, and orofacial clefting. A combined LOD score of 4.2 was obtained for the association between YAP1 loss-of-function mutations and the phenotype in these families. YAP1 encodes an effector of the HIPPO-pathway-induced growth response, and whole-mount in situ hybridization in mouse embryos has shown that Yap1 is strongly expressed in the eye, brain, and fusing facial processes. RT-PCR showed that an alternative transcription start site (TSS) in intron 1 of YAP1 and Yap1 is widely used in human and mouse development, respectively. Transcripts from the alternative TSS are predicted to initiate at codon Met179 relative to the canonical transcript (RefSeq NM_001130145). In these alternative transcripts, the c.370C>T mutation in family 1305 is within the 5' UTR and cannot result in nonsense-mediated decay (NMD). The c. 1066G>T mutation in family 132 should result in NMD in transcripts from either TSS. Amelioration of the phenotype by the alternative transcripts provides a plausible explanation for the phenotypic differences between the families.

Background Classic aniridia is a highly penetrant autosomal dominant disorder characterised by congenital absence of the iris, foveal hypoplasia, optic disc anomalies and progressive opacification of the cornea. >90% of cases of classic aniridia are caused by heterozygous, loss-of-function variants affecting the PAX6 locus. Methods Short-read whole genome sequencing was performed on 51 (39 affected) individuals from 37 different families who had screened negative for mutations in the PAX6 coding region. Results Likely causative mutations were identified in 22 out of 37 (59%) families. In 19 out of 22 families, the causative genomic changes have an interpretable deleterious impact on the PAX6 locus. Of these 19 families, 1 has a novel heterozygous PAX6 frameshift variant missed on previous screens, 4 have single nucleotide variants (SNVs) (one novel) affecting essential splice sites of PAX6 5' non-coding exons and 2 have deep intronic SNV (one novel) resulting in gain of a donor splice site. In 12 out of 19, the causative variants are large-scale structural variants; 5 have partial or whole gene deletions of PAX6, 3 have deletions encompassing critical PAX6 cis-regulatory elements, 2 have balanced inversions with disruptive breakpoints within the PAX6 locus and 2 have complex rearrangements disrupting PAX6. The remaining 3 of 22 families have deletions encompassing FOXC1 (a known cause of atypical aniridia). Seven of the causative variants occurred de novo and one cosegregated with familial aniridia. We were unable to establish inheritance status in the remaining probands. No plausibly causative SNVs were identified in PAX6 cis-regulatory elements. Conclusion Whole genome sequencing proves to be an effective diagnostic test in most individuals with previously unexplained aniridia. [ABSTRACT FROM AUTHOR]

PurposeTo describe the clinical findings of a patient with an early onset retinal dystrophy and a novel mutation in OTX2, and to compare these findings with previously reported cases.MethodsUsing direct sequencing, we screened 142 patients, who had either Leber congenital amaurosis (LCA) or early onset retinal dystrophy (EORD), for mutations in OTX2. All patients received a detailed ophthalmic examination including electroretinography and retinal imaging.ResultsOnly one mutation in OTX2 was identified. A novel heterozygous p.S138X stop mutation was identified in a seven-year-old male who had an infantile onset retinal dystrophy. The mutation was not present in either parent or in 181 blood donor samples. There was a history of failure to thrive in infancy, poor feeding, and growth hormone deficiency. Poor vision and nyctalopia was present from the first year. Funduscopy revealed a hyperpigmented peripapillary ring with a fine granular pigmentation of the RPE throughout the fundus. The scotopic bright flash ERG a-wave was subnormal and the waveform electronegative, in keeping with dysfunction both at the level of the photoreceptor and post-phototransduction. Visual function has been stable to date.ConclusionsMutations in OTX2 have been reported in association with major developmental malformations of the eye, with retinal dystrophies such as LCA, and with pituitary dysfunction and seizure activity in some cases. This case adds further support for a role of OTX2 both in retinal development and pituitary function, and highlights a novel retinal dystrophy phenotype seen in association with mutations in OTX2.

Pax6 is a transcription factor with an essential role in eye, central nervous system, and pancreas development. Its expression pattern is restricted to these specific domains within the developing embryo. Here four conserved elements are identified in Pax6 intron 7, showing a high level of sequence conservation between human, mouse, pufferfish, and zebrafish. Three of these are shown to act as c/s-regulatory elements, directing expression of a reporter gene to distinct subsets of the Pax6 expression domain. CE1 regulates gene expression in late eye development, CE2 drives expression in the diencephalon and in the developing heart tube where Pax6 is not normally expressed, while CE3 directs expression in rhombencephalon. CE2 is shown to be autoregulated in the diencephalon, responding to absence of Pax6. We identify a highly conserved Pax6 recognition site and demonstrate its ability to bind Pax6 specifically. CE1 is embedded in a CpG island, and we identify a novel Pax6 transcript which initiates from this region. Functional analysis of evolutionary conserved sequences pinpoints novel cis-acting elements that govern the regulation of the complex spatio-temporal and quantitative expression of Pax6.