Cat eye syndrome (CES) (MIM #115470), also known as Schmid-Fraccaro syndrome, is mainly caused by chromosome 22q11 inversion duplication or chromosome 22 partial tetrasomy (1) and is observed in 1:150,000 newborns. The major clinical features of CES are the combination of anal atresia, preauricular pits and tags, and/or iris coloboma (2). In addition, most patients also present additional malformations involving the cardiovascular and nervous systems, indicating a very wide clinical spectrum in terms of features and severity (3). Chromosome 22q11 is an ideal substrate for non-allelic homologous recombination (NAHR) and is highly susceptible to chromosomal rearrangements (4). Therefore, the 22q11 region is associated with many microduplication and microdeletion conditions such as CES, chromosome 22q11.2 microduplication syndrome (dup22q11) (MIM #608363), velocardiofacial syndrome (VCFS) (MIM #192430), and DiGeorge syndrome (DGS) (MIM #188400) (5, 6). Due to reciprocal rearrangements, 22q11.2 duplications and deletions are predicted to occur at the same frequency. However, the number of duplication cases, such as CES and dup22q11, reported are considerably less, which is likely because the duplications have a milder clinical effect. Affected dup22q11 individuals may have developmental delay, short stature, and facial abnormalities (7). The phenotypes of this condition like CES are highly variable, even among members of the same family. The chromosomal location and phenotype of CES are similar to those of dup22q11, which requires systematic differential diagnosis. Many eye malformations have been reported to be associated with CES, such as iris and chorioretinal coloboma, hypertelorism, and epicanthus. However, abnormality of eye movement is associated with Duane retraction syndrome (DURS) (MIM %126800, #604356 and #617041), congenital fibrosis of extraocular muscles (CFEOM) (MIM #135700, #602078, #600638, %609384, #616219), and abnormality of the optic nerve, as previously reported, and has not been associated with CES.

In the present study, we report on a Chinese family carrying a 22q11.1-q11.21 duplication of 1.7Mb tetrasomy (chr22:16,500,000–18,200,000, hg38) in two generations. The proband and her father presented with an abnormality of eye movement and were diagnosed with CES by ophthalmological examination, karyotyping, CNV-seq, and FISH. WES did not detect gene mutations associated with DURS, CFEOM, or abnormality of the optic nerve in the proband or father. Our findings broadened the symptom spectrum of CES syndrome, and abnormality of eye movement was noted in 22q11 for the first time. This laid the foundation for the pathogenesis, diagnostic targets, and drug research on the abnormality of eye movement.

CES covers a very wide spectrum of variable penetrance and expressivity, ranging from a normal phenotype to severe multisystemic symptoms. The three main characteristics are anal atresia, preauricular anomalies, and iris coloboma, but these characteristics are not consistently found in many patients with CES, as in our patients (2) (CES symptoms, Figure 4). The similarity of localization and symptoms makes the differential diagnosis between CES and dup22q11.2 difficult. A comparison of CES and dup22q11 symptoms with those of the proband and father showed that they suffered two very frequent phenotypes of CES (anal atresia and preauricular pits) and had a higher phenotype matching rate with CES than with dup22q11 (Supplementary Table S3) (for dup22q11 symptoms see Figure 4). According to the clinical manifestations of the proband and her father, the diagnosis of CES was made based on cytogenetic analysis and FISH showing the presence of a small supernumerary dicentric marker chromosome derived from chromosome 22q11. The CNV-seq results indicated that repeated regions included cat eye syndrome chromosome region, candidate 1 (CECR1), and candidate 2 (CECR2) genes located in a proximal 22q11 region, which further confirmed the previous diagnosis results.

In several previous reports, researchers found that abnormality of eye movement is associated with DURS, CFEOM, and abnormality of the optic nerve (8,9). There have been no reported cases of CES with an abnormality of eye movement. DURS was originally speculated to be the cause of this clinical condition. DURS is a congenital eye movement disorder with a prevalence of approximately 0.1% in the general population (9). DURS results in the restriction or absence of abduction, adduction, or both, and narrowing of the palpebral fissure and retraction of the globe on attempted adduction (10). CFEOM is part of a larger group of congenital cranial dysinnervation disorders and primarily affects ocular motility (8). However, the proband and her father had limited eye movement in the horizontal and vertical directions and had no features of retrograde eyeballs or palpebral fissure narrowing in attempted adduction, which excluded the possibility of CES combined with DURS. In addition, WES did not detect gene mutations associated with DURS, CFEOM, or abnormality of the optic nerve in the proband or father. Therefore, we think that the phenotype of eye movement disorder in the proband and the father is very likely related to the 1.7Mb tetrasomy. The 1.7Mb tetrasomy of the proband and her father on 22q11.1-q11.21 (16,500,000–18,200,000, hg38), which contains 11 OMIM morbid genes, namely, XKR3, IL17RA, CECR1 or ADA2, CECR2, SLC25A18, ATP6V1E1, BID, MICAL3, MIR648, PEX26, and TUBA8 (partially) (Supplementary Figure S1A). The proband and her father appeared to have a novel clinical symptom of eyeballs that could barely move. Compared with previously reported cases of CES and reviewing the function of the abovementioned genes (see Supplementary Table S4), BID and MICAL3 are most likely associated with this new symptom. BID encodes a cell death agonist and regulates apoptosis, and this gene plays a role in inducing premature cell death and can influence organ development or overall growth (11). MICAL3, a plexin signaling molecule expressed in motor neurons (12), may be directly associated with impaired ocular movement.

Although the karyotype of the proband’s grandparents was normal, the possibility of germline mosaicism could not be ruled out. In addition, non-homologous recombination also likely produced the proband’s father’s sSMC. Intra-genome duplicates greater than 1 kb in length and with at least 90% sequence similarity are termed low-copy repeats (LCRs) (4). When two homologous LCRs are less than 10 Mb apart from each other, they result in chromosomal or chromatid mislocations and unequal crossovers, resulting in the deletion or duplication of genomic segments between LCRs (13). Chromosome 22q11.2 harbors eight LCRs termed LCR22A-LCR22H, therefore, LCR22s are an ideal substrate for non-allelic homologous recombination (NAHR), resulting in rearrangements of 22q11.2 (Supplementary Figures S1B,C) (14). Normal chromosome 22 is most likely to generate a 1.7Mb tetrasomy on 22q11.1-q11.21 (16,500,000–18,200,000, hg38) by mismatch and NAHR. Before the formation of the father’s sperm, chromosomes replicate in spermatogonial cells, but the small marker chromosome does not. During the first meiosis of the primary spermatocyte and homologous chromosomal synapsis, the same two sequences of 1.7Mb in the small marker chromosome were paired to form the structure, as shown in the genetic model. Usually, only one of two centromeres is functional and is randomly pulled into a secondary spermatocyte by the spindle fiber. Therefore, the father had a 50% probability of transmitting the sSMC to the offspring (Supplementary Figure S1D).

In summary, CES exhibits a broad spectrum of clinical features, and many mildly affected patients probably remain undetected. Here, we report on a Chinese family carrying a 22q11.1-q11.21 duplication of 1.7Mb tetrasomy (16,500,000–18,200,000, hg38) in two generations. The proband and her father were diagnosed with CES by karyotype analyses, CNV-seq, and FISH, and presented an abnormality of eye movement, which was reported for the first time. No gene mutations associated with eye movement disorders, such as DURS and CFEOM, were detected in the peripheral blood of the proband and father by WES. Therefore, we think that the phenotype of eye movement disorder in the proband and her father is very likely related to BID and MICAL3 in the 1.7Mb tetrasomy. Our findings broadened the symptom spectrum of CES syndrome, and, for the first time, mutations causing abnormality of eye movement were located at 22q11. This laid the foundation for the pathogenesis, diagnostic targets, and drug research in abnormality of eye movement and was helpful for the early diagnosis and intervention of CES.