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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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Purpose: Most classical aniridia is caused by PAX6 haploinsufficiency. PAX6 missense variants can be hypomorphic or mimic haploinsufficiency. We hypothesized that missense variants also cause previously undescribed disease by altering the affinity and/or specificity of PAX6 genomic interactions., Methods: We screened PAX6 in 372 individuals with bilateral microphthalmia, anophthalmia, or coloboma (MAC) from the Medical Research Council Human Genetics Unit eye malformation cohort (HGU eye ) and reviewed data from the Deciphering Developmental Disorders study. We performed cluster analysis on PAX6-associated ocular phenotypes by variant type and molecular modeling of the structural impact of 86 different PAX6 causative missense variants., Results: Eight different PAX6 missense variants were identified in 17 individuals (15 families) with MAC, accounting for 4% (15/372) of our cohort. Seven altered the paired domain (p.[Arg26Gln]x1, p.[Gly36Val]x1, p.[Arg38Trp]x2, p.[Arg38Gln]x1, p.[Gly51Arg]x2, p.[Ser54Arg]x2, p.[Asn124Lys]x5) and one the homeodomain (p.[Asn260Tyr]x1). p.Ser54Arg and p.Asn124Lys were exclusively associated with severe bilateral microphthalmia. MAC-associated variants were predicted to alter but not ablate DNA interaction, consistent with the electrophoretic mobility shifts observed using mutant paired domains with well-characterized PAX6-binding sites. We found no strong evidence for novel PAX6-associated extraocular disease., Conclusion: Altering the affinity and specificity of PAX6-binding genome-wide provides a plausible mechanism for the worse-than-null effects of MAC-associated missense variants.

Importance: We describe novel ocular phenotypic features caused by mutations in ADAMTS18. The exact role of ADAMTS18 in ocular disease is unclear, and our work further contributes to the understanding of this gene and its protein., Objective: To expand the phenotypic characterization in patients with homozygous mutations in ADAMTS18 and report novel mutational data., Design, Setting, and Participants: A case series with genetic investigations was conducted at tertiary referral clinical and university settings. Three families participated., Main Outcome Measures: Phenotype and genotype description of 3 families., Results: Four affected patients from 3 families with an unusual ocular phenotype had full ophthalmic and systemic examination. A single affected individual in the first family had bilateral microcornea, ectopic pupils, and cone-rod dystrophy. In a second family, 2 brothers showed bilateral microcornea, childhood cataract, ectopia lentis, rhegmatogenous retinal detachment, and cone-rod dystrophy. In the third family, a single affected individual had the same features as those in family 2, without ectopia lentis. Causative mutations were sought using homozygosity mapping, Sanger sequencing, and massively parallel sequencing of the whole exome. Novel homozygous mutations in ADAMTS18 were identified, consisting of c.1067T>A [p.L356*] in the first proband, c.2159G>C [p.C720S] in the 2 affected brothers, and c.1952G>A [p.R651Q] in the third proband. All 3 mutations are predicted to be pathogenic., Conclusions and Relevance: Mutations in ADAMTS18 are associated with ocular developmental abnormalities including microcornea, ectopia lentis, and early onset of cone-rod dystrophy. This report provides further evidence that ADAMTS18 plays a key role in ocular development. Physicians should consider screening ADAMTS18 in patients with microcornea and cone-rod dystrophy.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

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., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing., Competing Interests: The authors have declared that no competing interests exist.

Major malformations of the human eye, including microphthalmia and anophthalmia, are examples of phenotypes that recur in families yet often show no clear Mendelian inheritance pattern. Defining loci by mapping is therefore rarely feasible. Using a candidate-gene approach, we have identified heterozygous coding-region changes in the homeobox gene OTX2 in eight families with ocular malformations. The expression pattern of OTX2 in human embryos is consistent with the eye phenotypes observed in the patients, which range from bilateral anophthalmia to retinal defects resembling Leber congenital amaurosis and pigmentary retinopathy. Magnetic resonance imaging scans revealed defects of the optic nerve, optic chiasm, and, in some cases, brain. In two families, the mutations appear to have occurred de novo in severely affected offspring, and, in two other families, the mutations have been inherited from a gonosomal mosaic parent. Data from these four families support a simple model in which OTX2 heterozygous loss-of-function mutations cause ocular malformations. Four additional families display complex inheritance patterns, suggesting that OTX2 mutations alone may not lead to consistent phenotypes. The high incidence of mosaicism and the reduced penetrance have implications for genetic counseling.

Microphthalmia, anophthalmia and coloboma (MAC) are distinct phenotypes that represent a continuum of structural developmental eye defects. In severe bilateral cases (anophthalmia or severe microphthalmia) the genetic cause is now identifiable in approximately 80 percent of cases, with de novo heterozygous loss-of-function mutations in SOX2 or OTX2 being the most common. The genetic cause of other forms of MAC, in particular isolated coloboma, remains unknown in the majority of cases. This review will focus on MAC phenotypes that are associated with mutation of the genes SOX2, OTX2, PAX6, STRA6, ALDH1A3, RARB, VSX2, RAX, FOXE3, BMP4, BMP7, GDF3, GDF6, ABCB6, ATOH7, C12orf57, TENM3 (ODZ3), and VAX1. Recently reported mutation of the SALL2 and YAP1 genes are discussed in brief. Clinical and genetic features were reviewed in a total of 283 unrelated MAC cases or families that were mutation-positive from these 20 genes. Both the relative frequency of mutations in MAC cohort screens and the level of confidence in the assignment of disease-causing status were evaluated for each gene. [ABSTRACT FROM AUTHOR]

A girl with aniridia, microphthalmia, microcephaly and café au lait macules was found to have mutations in PAX6, NF1 and OTX2. A novel PAX6 missense mutation (p.R38W) was inherited from her mother whose iris phenotype had not been evident because of ocular neurofibromatosis. Analysis of the NF1 gene in the proband, prompted by the mother's diagnosis and the presence of café au lait spots, revealed a nonsense mutation (p.R192X). Subsequently an OTX2 nonsense mutation (p.Y179X) was identified and shown to be inherited from her father who was initially diagnosed with Leber's congenital amaurosis. Since individual mutations in PAX6, OTX2 or NF1 can cause a variety of severe developmental defects, the proband's phenotype is surprisingly mild. This case shows that patients with complex phenotypes should not be eliminated from subsequent mutation analysis after one or even two mutations are found.European Journal of Human Genetics (2007) 15, 898–901; doi:10.1038/sj.ejhg.5201826; published online 4 April 2007 [ABSTRACT FROM AUTHOR]

A submicroscopic deletion containing SOX2 was identified at the 3q breakpoint in a child with t(3;11)(q26.3;p11.2) associated with bilateral anophthalmia. Subsequent SOX2 mutation analysis identified de novo truncating mutations ofSOX2 in 4 of 35 (11%) individuals with anophthalmia. Both eyes were affected in all cases with an identified mutation. [ABSTRACT FROM AUTHOR]

We observed two unrelated consanguineous families with malformation syndromes sharing anophthalmia and distinct eyebrows as common signs, but differing for alveolar capillary dysplasia or complex congenital heart defect in one and diaphragmatic hernia in the other family. Homozygosity mapping revealed linkage to a common locus on chromosome 15, and pathogenic homozygous mutations were identified in STRA6, a member of a large group of ‘stimulated by retinoic acid’ genes encoding novel transmembrane proteins, transcription factors, and secreted signaling molecules or proteins of largely unknown function. Subsequently, homozygous STRA6 mutations were also demonstrated in 3 of 13 patients chosen on the basis of significant phenotypic overlap to the original cases. While a homozygous deletion generating a premature stop codon (p.G50AfsX22) led to absence of the immunoreactive protein in patient's fibroblast culture, structural analysis of three missense mutations (P90L, P293L, and T321P) suggested significant effects on the geometry of the loops connecting the transmembrane helices of STRA6. Two further variations in the C-terminus (T644M and R655C) alter specific functional sites, an SH2-binding motif and a phosphorylation site, respectively. STRA6 mutations thus define a pleiotropic malformation syndrome representing the first human phenotype associated with mutations in a gene from the ‘STRA’ group. [ABSTRACT FROM AUTHOR]