Following informed consent, we obtained pedigree information, clinical data, and blood samples from the families. We obtained approval for human subject research from the ethics committee of the Children's Hospital of Nanjing Medical University.

Trio-based WES was performed as previously described (11). In brief, genomic DNA was isolated from blood lymphocytes using the DNA isolation kit (Tiangen, China). Genomic DNA was sheared into fragments and then hybridized with the xGen Exome Research Panel v1.0 probe sequence capture array from IDT (Integrated Device Technology, United States) to enrich the exonic region. The enriched libraries were analyzed on an Illumina HiSeq XTen (Illumina, United States) platform. Low-quality variations of the quality score <20 (Q20) were filtered out. Sequencing reads were mapped to the GRCh37/Hg19 reference genome via Burrows-Wheeler Aligner (BWA) software. Single nucleotide variation (SNV) and inserts and deletions (INDEL) were filtered using GATK software (https://software.broadinstitute.org/gatk/). All identified variants were filtered using the 1000 Genomes Project (Chinese), dbSNP, Genome Aggregation Database (gnomAD), and ExAC database. Variants with a minor allele frequency higher than 5% were filtered out. Finally, the candidate variants were evaluated using the ACMG (American College of Medical Genetics and Genomics) criteria and further validated by direct Sanger sequencing.

The two heterozygous TENM3 variants in family 1 were both located in exon 19. To obtain a clean Sanger sequence of the two heterozygous variants, we cloned 383 bp-long PCR products of TENM3 exon19 using the pCR2.1-TOPO plasmid vector system (Invitrogen). PCR products were generated using TENM3 forward primer 5-ATCCTCAGCGTCAGGCAAGGAA-3 and reverse primer 5-TCCCCTGTCCCTGCGACGAC-3. The TA clone sequencing was conducted as previously described (12).

Protein domain structure depictions and evaluation were based on the UniProt (Universal Protein Resource) database. Orthologous proteins used to evaluate evolutionary conservation were obtained from the Ensemble Genome Browser and were aligned using the Clustal Omega multiple sequence alignment tool (EMBL-EBI). And we evaluated the crystal structure of the two missense TENM3 variants in patient 2 using the online server, UCSF ChimeraX (http://www.cgl.ucsf.edu/chimerax//).

Patient 1 was the 5-month-old daughter of Chinese non-consanguineous parents. She had two older brothers and both are healthy. She was born at 39 weeks of gestation. Her birth weight was 2.7 kg (−1.46 SD) and her length was 40 cm (−5.89 SD). At birth, she was diagnosed with bilateral microphthalmia and congenital cataract and she appeared to have pendular nystagmus and esotropia (Figure 1B). She first visited our department of rehabilitation at the age of 5 months for developmental delay. Her developmental milestones were delayed. Her head control was unstable and she was unable to turn over and grab the toy on her chest. On careful physical examination, her height, weight, and head circumference were 57 cm (−4.03 SD), 5 kg (−3.75 SD), and 37 cm (−4.28 SD), respectively. Her prominent and low-set ears were noted (Figure 1B). Fundus examination revealed the posterior pole of the retina colobomas involving the optic discs and the fovea (Figure 1C). Her hearing assessment was normal. According to the Gesell Developmental Diagnostic Scale for children, the proband's gross motor skills indicated a developmental age of 8 weeks; the fine motor skills indicated a developmental age of 8 weeks; Her blood counts, liver and renal function tests, thyroid profile, and metabolic screen by mass spectrometry were normal. Brain magnetic resonance imaging (MRI) showed no structure malformations except a widening in the frontotemporal extracerebral space. Her mother had left exotropia and graduated from middle school with poor grades.

Patient 2 was the 3-year-and-5-month-old son of Chinese non-consanguineous parents. His weight was 17 kg (0.8 SD) and his height was 101 cm (0.2 SD). At birth, it was noted that he had bilateral esotropia. At the age of 3 years old, his bilateral esotropia was resolved (Figure 2B). What's more, his anterior segment and fundus examination showed no structural anomalies (Figure 2C). He had astigmatism in both eyes (−1.25 DC). He had mild motor delay. He was able to walk without support at 17 months. He had significant speech delay, not producing any meaningful words at 2 years and 5 months and speaking a few simple words (about 10 words) at 3 years and 5 months. He showed poor eye contact and was not interested in his surroundings, so his social interaction was abnormal. According to the Gesell Developmental Diagnostic Scale for children, the proband's delayed speech indicated a developmental age of only 18 months. He was also evaluated by the Autistic Behavior Checklist (ABC) and Childhood Autism Rating Scale (CARS) (ABC: score 40; CARS: score 32). His blood counts, liver and renal function tests, thyroid profile, and metabolic screen by mass spectrometry were normal. His hearing evaluation was also normal. His prominent and big ears were noted. A brain MRI did not show any intracranial abnormalities. His father had no abnormal eye appearance, but his eyes were myopic (−7.00 DS), and his mother was healthy. His grandfather had a history of fundus abnormality.

Initial genetic testing for patient 1 was carried out via WES, revealing two heterozygous variants in the TENM3 gene (Genbank association number: NM_001080477): c.3847_3855del; p.Leu1283_Ser1285del and c.3698_3699insA; p.Thr1233Thrfs*20 (Figure 1A). Sanger sequencing of the parents confirmed that the variant p.Leu1283_Ser1285del was inherited maternally. The variant p.Thr1233Thrfs*20 was confirmed to be de novo. The two variants were both located in exon 19. A TA clone sequencing including the fragment of exon 19 demonstrated that the two variants occurred biallelically (Figure 1D). The two variants were absent from the control database gnomAD. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the variant c.3698_3699insA; p.Thr1233Thrfs*20 can be categorized as pathogenic (PVS1 + PM2 + PP4) and the variant c.3847_3855del; p.Leu1283_Ser1285del can be categorized as a variant of likely pathogenic (PM2 + PM3 + PM4).

Similarly, patient 2 had a WES that revealed compound heterozygous variants of the TENM3 gene, c.941C > T; p.Ala314Val and c.6464T > C; p.Leu2155Pro (Figure 2A). Sanger sequencing confirmed that his mother carried the c.941C > T (p.Ala314Val) mutation and his father carried the c.6464T > C (p.Leu2155Pro) mutation (Figure 2D). The p.Ala314Val variant was absent from the gnomAD. The p.Leu2155Pro variant occurred once heterozygously in the gnomAD. Both the missense changes yielded predominantly deleterious prediction scores using five algorithms (Polyphen2_HDIV, MutationTaster, SIFT, Provean, and REVEL); the predicted results can be found in the Supplementary Material.

The Ala314 change was located in the transmembrane domain and was evolutionarily well-conserved from Homo sapiens to zebrafish (Figures 3A,B). The Leu2155 residue was located in the YD-repeats domain and was well-conserved to zebrafish as well (Figures 3A,B). According to the ACMG guidelines, both variants, c.941C > T; p.Ala314Val and c.6464T > C; p.Leu2155Pro, can be categorized as variants of unknown significance (PM2 + PP2 + PP3). In evaluating the deleteriousness of the two missense variants in patient 2, three-dimensional structural modeling of the TENM3 protein showed that the mutations did not change the hydrogen bonding in the protein (blue), but repulsive force (purple) was generated between the R group of amino acid side chain and other nearby groups, which is unfavorable to the folding of the active protein and results in protein conformational instability (Figure 4).