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Microphthalmia, Anophthalmia and Coloboma (MAC) form a spectrum of congenital eye malformations responsible for severe visual impairment. Despite the exploration of hundreds of genes by High-Throughput Sequencing (HTS), most of the patients remain without genetic diagnosis. One explanation could be the not yet demonstrated involvement of somatic mosaicism (undetected by conventional analysis pipelines) in those patients. Furthermore, the proportion of parental germline mosaicism in presumed de novo variations is still unknown in ocular malformations. Thus, using dedicated bioinformatics pipeline designed to detect mosaic variants, we reanalysed the sequencing data obtained from a 119 ocular development genes panel performed on blood samples of 78 probands with sporadic MAC without genetic diagnosis. Using the same HTS strategy, we sequenced 80 asymptomatic parents of 41 probands carrying a disease-causing variant in an ocular development gene considered de novo after Sanger sequencing of both parents. Reanalysis of the previously sequencing data did not find any mosaic variant in probands without genetic diagnosis. However, HTS of parents revealed undetected SOX2and PAX6mosaic variants in two parents. Finally, this work, performed on two large cohorts of patients with MAC spectrum, provides for the first time an overview of the interest of looking for mosaicism in ocular development disorders. Somatic mosaicism does not appear to be frequent in MAC spectrum and might explain only few diagnoses. Thus, other approaches such as whole genome sequencing should be considered in those patients. Parental mosaicism is however not that rare (around 5%) and challenging for genetic counselling.

Biallelic pathogenic variants in ALDH1A3are 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 ALDH1A3variants remains unclear. Here, we describe seven unrelated families with biallelic pathogenic ALDH1A3variants: 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 ALDH1A3variants 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 ALDH1A3variants in nonconsanguineous families with A/M.

Defects in optic fissure closure can lead to congenital ocular coloboma. This ocular malformation, often associated with microphthalmia, is described in various clinical forms with different inheritance patterns and genetic heterogeneity. In recent times, the identification of an increased number of genes involved in numerous cellular functions has led to a better understanding in optic fissure closure mechanisms. Nevertheless, most of these genes are also involved in wider eye growth defects such as micro-anophthalmia, questioning the mechanisms controlling both extension and severity of optic fissure closure defects. However, some genes, such as FZD5, have only been so far identified in isolated coloboma. Thus, to estimate the frequency of implication of different ocular genes, we screened a cohort of 50 patients affected by ocular coloboma by using targeted sequencing of 119 genes involved in ocular development. This analysis revealed seven heterozygous (likely) pathogenic variants in RARB, MAB21L2, RBP4, TFAP2A,and FZD5. Surprisingly, three out of the seven variants detected herein were novel disease-causing variants in FZD5identified in three unrelated families with dominant inheritance. Although molecular diagnosis rate remains relatively low in patients with ocular coloboma (14% (7/50) in this work), these results, however, highlight the importance of genetic screening, especially of FZD5, in such patients. Indeed, in our series, FZD5variants represent half of the genetic causes, constituting 6% (3/50) of the patients who benefited from a molecular diagnosis. Our findings support the involvement of FZD5in ocular coloboma and provide clues for screening this gene during current diagnostic procedures.

Heterozygous COL2A1 variants cause a wide spectrum of skeletal dysplasia termed type II collagenopathies. We assessed the impact of this gene in our French series. A decision tree was applied to select 136 probands (71 Stickler cases, 21 Spondyloepiphyseal dysplasia congenita cases, 11 Kniest dysplasia cases, and 34 other dysplasia cases) before molecular diagnosis by Sanger sequencing. We identified 66 different variants among the 71 positive patients. Among those patients, 18 belonged to multiplex families and 53 were sporadic. Most variants (38/44, 86%) were located in the triple helical domain of the collagen chain and glycine substitutions were mainly observed in severe phenotypes, whereas arginine to cysteine changes were more often encountered in moderate phenotypes. This series of skeletal dysplasia is one of the largest reported so far, adding 44 novel variants (15%) to published data. We have confirmed that about half of our Stickler patients (46%) carried a COL2A1 variant, and that the molecular spectrum was different across the phenotypes. To further address the question of genotype–phenotype correlation, we plan to screen our patients for other candidate genes using a targeted next-generation sequencing approach.

Verheij syndrome, also called 8q24.3 microdeletion syndrome, is a rare condition characterized by ante- and postnatal growth retardation, microcephaly, vertebral anomalies, joint laxity/dislocation, developmental delay (DD), cardiac and renal defects and dysmorphic features. Recently, PUF60 (Poly-U Binding Splicing Factor 60 kDa), which encodes a component of the spliceosome, has been discussed as the best candidate gene for the Verheij syndrome phenotype, regarding the cardiac and short stature phenotype. To date, only one patient has been reported with a de novo variant in PUF60 that probably affects function (c.505C>T leading to p.(His169Tyr)) associated with DD, microcephaly, craniofacial and cardiac defects. Additional patients were required to confirm the pathogenesis of this association and further delineate the clinical spectrum. Here we report five patients with de novo heterozygous variants in PUF60 identified using whole exome sequencing. Variants included a splice-site variant (c.24+1G>C), a frameshift variant (p.(Ile136Thrfs*31)), two nonsense variants (p.(Arg448*) and p.(Lys301*)) and a missense change (p.(Val483Ala)). All six patients with a PUF60 variant (the five patients of the present study and the unique reported patient) have the same core facial gestalt as 8q24.3 microdeletions patients, associated with DD. Other findings include feeding difficulties (3/6), cardiac defects (5/6), short stature (5/6), joint laxity and/or dislocation (5/6), vertebral anomalies (3/6), bilateral microphthalmia and irido–retinal coloboma (1/6), bilateral optic nerve hypoplasia (2/6), renal anomalies (2/6) and branchial arch defects (2/6). These results confirm that PUF60 is a major driver for the developmental, craniofacial, skeletal and cardiac phenotypes associated with the 8q24.3 microdeletion.