Biallelic variants in the small optic lobe calpain CAPN15 are associated with congenital eye anomalies, deafness and other neurodevelopmental deficits.

Microphthalmia, coloboma and cataract are part of a spectrum of developmental eye disorders in humans affecting ~12 per 100 000 live births. Currently, variants in over 100 genes are known to underlie these conditions. However, at least 40% of affected individuals remain without a clinical genetic diagnosis, suggesting variants in additional genes may be responsible. Calpain 15 (CAPN15) is an intracellular cysteine protease belonging to the non-classical small optic lobe (SOL) family of calpains, an important class of developmental proteins, as yet uncharacterized in vertebrates. We identified five individuals with microphthalmia and/or coloboma from four independent families carrying homozygous or compound heterozygous predicted damaging variants in CAPN15. Several individuals had additional phenotypes including growth deficits, developmental delay and hearing loss. We generated Capn15 knockout mice that exhibited similar severe developmental eye defects, including anophthalmia, microphthalmia and cataract, and diminished growth. We demonstrate widespread Capn15 expression throughout the brain and central nervous system, strongest during early development, and decreasing postnatally. Together, these findings demonstrate a critical role of CAPN15 in vertebrate developmental eye disorders, and may signify a new developmental pathway.

De Novo Missense Variants in FBXW11 Cause Diverse Developmental Phenotypes Including Brain, Eye, and Digit Anomalies.

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include ß-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw.

NAA10 polyadenylation signal variants cause syndromic microphthalmia.

BACKGROUND: A single variant in NAA10 (c.471+2T>A), the gene encoding N-acetyltransferase 10, has been associated with Lenz microphthalmia syndrome. In this study, we aimed to identify causative variants in families with syndromic X-linked microphthalmia. METHODS: Three families, including 15 affected individuals with syndromic X-linked microphthalmia, underwent analyses including linkage analysis, exome sequencing and targeted gene sequencing. The consequences of two identified variants in NAA10 were evaluated using quantitative PCR and RNAseq. RESULTS: Genetic linkage analysis in family 1 supported a candidate region on Xq27-q28, which included NAA10. Exome sequencing identified a hemizygous NAA10 polyadenylation signal (PAS) variant, chrX:153,195,397T>C, c.*43A>G, which segregated with the disease. Targeted sequencing of affected males from families 2 and 3 identified distinct NAA10 PAS variants, chrX:g.153,195,401T>C, c.*39A>G and chrX:g.153,195,400T>C, c.*40A>G. All three variants were absent from gnomAD. Quantitative PCR and RNAseq showed reduced NAA10 mRNA levels and abnormal 3' UTRs in affected individuals. Targeted sequencing of NAA10 in 376 additional affected individuals failed to identify variants in the PAS. CONCLUSION: These data show that PAS variants are the most common variant type in NAA10-associated syndromic microphthalmia, suggesting reduced RNA is the molecular mechanism by which these alterations cause microphthalmia/anophthalmia. We reviewed recognised variants in PAS associated with Mendelian disorders and identified only 23 others, indicating that NAA10 harbours more than 10% of all known PAS variants. We hypothesise that PAS in other genes harbour unrecognised pathogenic variants associated with Mendelian disorders. The systematic interrogation of PAS could improve genetic testing yields.

Genetics of anophthalmia and microphthalmia. Part 1: Non-syndromic anophthalmia/microphthalmia.

Eye formation is the result of coordinated induction and differentiation processes during embryogenesis. Disruption of any one of these events has the potential to cause ocular growth and structural defects, such as anophthalmia and microphthalmia (A/M). A/M can be isolated or occur with systemic anomalies, when they may form part of a recognizable syndrome. Their etiology includes genetic and environmental factors; several hundred genes involved in ocular development have been identified in humans or animal models. In humans, around 30 genes have been repeatedly implicated in A/M families, although many other genes have been described in single cases or families, and some genetic syndromes include eye anomalies occasionally as part of a wider phenotype. As a result of this broad genetic heterogeneity, with one or two notable exceptions, each gene explains only a small percentage of cases. Given the overlapping phenotypes, these genes can be most efficiently tested on panels or by whole exome/genome sequencing for the purposes of molecular diagnosis. However, despite whole exome/genome testing more than half of patients currently remain without a molecular diagnosis. The proportion of undiagnosed cases is even higher in those individuals with unilateral or milder phenotypes. Furthermore, even when a strong gene candidate is available for a patient, issues of incomplete penetrance and germinal mosaicism make diagnosis and genetic counseling challenging. In this review, we present the main genes implicated in non-syndromic human A/M phenotypes and, for practical purposes, classify them according to the most frequent or predominant phenotype each is associated with. Our intention is that this will allow clinicians to rank and prioritize their molecular analyses and interpretations according to the phenotypes of their patients.

New GJA8 variants and phenotypes highlight its critical role in a broad spectrum of eye anomalies.

GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype-phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development.

Further delineation of an entity caused by CREBBP and EP300 mutations but not resembling Rubinstein-Taybi syndrome.

In 2016, we described that missense variants in parts of exons 30 and 31 of CREBBP can cause a phenotype that differs from Rubinstein-Taybi syndrome (RSTS). Here we report on another 11 patients with variants in this region of CREBBP (between bp 5,128 and 5,614) and two with variants in the homologous region of EP300. None of the patients show characteristics typical for RSTS. The variants were detected by exome sequencing using a panel for intellectual disability in all but one individual, in whom Sanger sequencing was performed upon clinical recognition of the entity. The main characteristics of the patients are developmental delay (90%), autistic behavior (65%), short stature (42%), and microcephaly (43%). Medical problems include feeding problems (75%), vision (50%), and hearing (54%) impairments, recurrent upper airway infections (42%), and epilepsy (21%). Major malformations are less common except for cryptorchidism (46% of males), and cerebral anomalies (70%). Individuals with variants between bp 5,595 and 5,614 of CREBBP show a specific phenotype (ptosis, telecanthi, short and upslanted palpebral fissures, depressed nasal ridge, short nose, anteverted nares, short columella, and long philtrum). 3D face shape demonstrated resemblance to individuals with a duplication of 16p13.3 (the region that includes CREBBP), possibly indicating a gain of function. The other affected individuals show a less specific phenotype. We conclude that there is now more firm evidence that variants in these specific regions of CREBBP and EP300 result in a phenotype that differs from RSTS, and that this phenotype may be heterogeneous.

Mutations in SOX2 cause anophthalmia.

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 of SOX2 in 4 of 35 (11%) individuals with anophthalmia. Both eyes were affected in all cases with an identified mutation.

Analysis of Fibroblast Growth Factor 14 (FGF14) structural variants reveals the genetic basis of the early onset nystagmus locus NYS4 and variable ataxia.

Nystagmus (involuntary, rhythmical eye movements) can arise due to sensory eye defects, in association with neurological disorders or as an isolated condition. We identified a family with early onset nystagmus and additional neurological features carrying a partial duplication of FGF14, a gene associated with spinocerebellar ataxia type 27 (SCA27) and episodic ataxia. Detailed eye movement analysis revealed oculomotor anomalies strikingly similar to those reported in a previously described four-generation family with early onset nystagmus and linkage to a region on chromosome 13q31.3-q33.1 (NYS4). Since FGF14 lies within NYS4, we revisited the original pedigree using whole genome sequencing, identifying a 161 kb heterozygous deletion disrupting FGF14 and ITGBL1 in the affected individuals, suggesting an FGF14-related condition. Therefore, our study reveals the genetic variant underlying NYS4, expands the spectrum of pathogenic FGF14 variants, and highlights the importance of screening FGF14 in apparently isolated early onset nystagmus.

Clinical and genetic analysis further delineates the phenotypic spectrum of ALDH1A3-related anophthalmia and microphthalmia.

Biallelic pathogenic variants in ALDH1A3 are responsible for approximately 11% of recessively inherited cases of severe developmental eye anomalies. Some individuals can display variable neurodevelopmental features, but the relationship to the ALDH1A3 variants remains unclear. Here, we describe seven unrelated families with biallelic pathogenic ALDH1A3 variants: four compound heterozygous and three homozygous. All affected individuals had bilateral anophthalmia/microphthalmia (A/M), three with additional intellectual or developmental delay, one with autism and seizures and three with facial dysmorphic features. This study confirms that individuals with biallelic pathogenic ALDH1A3 variants consistently manifest A/M, but additionally display neurodevelopmental features with significant intra- and interfamilial variability. Furthermore, we describe the first case with cataract and highlight the importance of screening ALDH1A3 variants in nonconsanguineous families with A/M.

Individuals with heterozygous variants in the Wnt-signalling pathway gene FZD5 delineate a phenotype characterized by isolated coloboma and variable expressivity.

BACKGROUND: Anophthalmia, microphthalmia and coloboma are a genetically heterogenous spectrum of developmental eye disorders. Recently, variants in the Wnt-pathway gene Frizzled Class Receptor 5 (FZD5) have been identified in individuals with coloboma and rarely microphthalmia, sometimes with additional phenotypes and variable penetrance. MATERIALS AND METHODS: We identified variants in FZD5 in individuals with developmental eye disorders from the UK (including the DDD Study [www.ddduk.org/access.html]), France and Spain using whole genome/exome sequencing or customized NGS panels of ocular development genes. RESULTS: We report eight new families with FZD5 variants and ocular coloboma. Three individuals presented with additional syndromic features, two explicable by additional variants in other genes (SLC12A2 and DDX3X). In two families initially showing incomplete penetrance, re-examination of apparently unaffected carrier individuals revealed subtle ocular colobomatous phenotypes. Finally, we report two families with microphthalmia in addition to coloboma, representing the second and third reported cases of this phenotype in conjunction with FZD5 variants. CONCLUSIONS: Our findings indicate FZD5 variants are typically associated with isolated ocular coloboma, occasionally microphthalmia, and that extraocular phenotypes are likely to be explained by other gene alterations.