In this study, we detected potentially causative variants in 42% of probands with ASD, which is higher than the reported proportion, which ranges from 10–25% . Table 3 summarizes the characteristics of different genetic studies on ASD. Potential explanations for a higher detection rate of causative variants in our cohort are ethnicities studied, differences in case selection, the number of genes analyzed, and sample size. Our cohort consisted of a unique demographic from South Florida, including large Hispanic and Caribbean populations. Earlier studies focused on European, Asian, African, and Middle Eastern populations [27, 28]. We did not identify recurrent variants enriched in our cohort; the difference between ethnicities of our cohort and those of earlier studies does not appear to explain our higher detection rate. In our cohort, families with Hispanic ancestry appear to have a higher detection rate (Hispanic vs. Non-Hispanic is 7/12 vs 3/12). Additionally, the difference between whites and blacks is noticable: only 1 out of 7 black families is solved while 9 of 17 white families studied are found to have potentailly causative variants. Majority of our black families were from the Caribbean, suggesting that the underlying genetic factors of ASD in the Caribbean remain largely unknown. Another important difference between our study and previous studies is the spectrum of ASD being analyzed. We looked at a wide range of ASD conditions, such as Peters anomaly, aniridia, AR syndrome, and PCG. Some other studies focused on a specific phenotype, such as Peters anomaly  or primary open-angle glaucoma/primary angle-closure glaucoma . Recognized gene variants for some focused phenotypes are present in smaller portions of affected individauls, which likely contributes to higher detection rate in our study. We used ES to cover all genes previously associated with ASD and some previous studies used gene panels, which may not include all associated genes. While targeted next-generation sequencing gene panels potentially provide higher coverage for individual genes and lower cost, ES as a research tool reduces the need of development and validation of custom panels. Finally, our cohort is smaller in size compared to previous cohorts and may have a higher detection rate by chance.
Causative variant detection in published studies.
ES or Gene Panel
Causative Variants Detected in ASD
Weh et al. 
Syndromic Peters anomaly: 20
Isolated Peters anomaly: 7
Children’s Hospital of Wisconsin (USA)
Population subtypes were not mentioned
Huang et al. 
ES of 43 genes associated with ASD, microcornea or microphthalmia
Patel et al. 
MAC: 98 cases
ASDA: 113 cases
Other or syndromic: 8 cases
RET: 49 cases
Congenital cataracts and lens- associated conditions: 9 cases
White European: 139
South Asian: 21
Black African: 7
Arabic or Middle Eastern: 5
Black Caribbean: 2
Oculome panel of 429 known eye disease genes
Congenital cataracts and lens-associated conditions: 88.9%
Other or syndromic: 37.5%
Peters anomaly: 8
Congenital corneal dystrophy: 1
Microphthalmia with glaucoma: 1
White, Hispanic: 11 (7 solved)
Black, Hispanic: 1 (0 solved)
White, non-Hispanic: 6 (2 solved)
Black, non-Hispanic: 6 (1 solved)
ES of 92 genes associated with eye phenotypes
Peters anomaly: 75%
|ASD: Anterior segment dysgenesis, ASDA: Anterior segment developmental anomalies including glaucoma, ES: exome sequencing, MAC: Microphthalmia-anophthalmia-coloboma, PACG: Primary angle-closure glaucoma, POAG: Primary open-angle glaucoma, RET: Retinal dystrophies|
Identified variants in PAX6, FOXC1, TP63, BMP4, B3GLCT, and GJA1 are considered likely pathogenic or pathogenic based on ACMG guidelines. One proband with Peters anomaly was heterozygous for two VUSs in PXDN. One variant is a change from leucine to proline in position 1274. This variant affects a conserved residue and is predicted to affect protein function with a Rare Exome Ensemble Learner (REVEL) score of 0.776, which is a combination of 13 indivudal tools for pathogeneticity prediction of missense variants . The second variant shows a change from serine to leucine in position 759. This missense variant is also predicted to make an impact on protein function with a REVEL score of 0.8399 Each parent is heterozygous for one variant suggesting that these two variants are in trans (Fig. 1). Biallelic PXDN variants have been reported with various eye anomalies including microphthalmia, congenital cataracts, microcornea, sclerocornea, and glaucoma [30, 31]. Therefore, it is possible that the identified variants are the cause of Peters anomaly in our patient. Similarly, one proband was homozygous for the FOXE3 p.I97M variant, which is a VUS. The allele frequency of this variant on gnomAD is 0.00002015. Multiple in-silico prediction tools show a damaging effect. This variant has been previously reported in a case with ASD . Therefore we consider the FOXE3 variant a likely cause of the eye phenotype in our proband.
The observed phenotypes in the 10 probands and the variants identified are generally consistent with prior studies. However, in family 5, the proband is heterozygous for a TP63 gene variant. Typically, TP63 mutations have been reported in Rapp-Hodgkin (MIM 129400), ADULT (Acro-Dermato-Ungual-Lacrimal-Tooth; MIM 103285), EEC (Ectrodactyly-Ectodermal dysplasia-Cleft lip/palate; MIM 604292), Hay-Wells (MIM 106260), Limb-mammary (MIM 603543), and split hand/foot malformation (MIM 605289) syndromes. In addition to various systemic anomalies, eye findings of these syndromes include blue irides, photophobia, blepharophimosis, blepharitis, dacryocystitis, and lacrimal duct abnormalities [32–37]. Our proband was diagnosed with Peters anomaly in the right eye as well as with syndactyly of third and fourth toes in the left foot, vesicoureteral reflux, cleft lip and palate, possible glaucoma, and nasolacrimal abnormalities. All of these findings except for Peters anomaly have been reported in patients with TP63 variants. Another TP63 variant (p.R343W) affecting the same amino acid residue has been reported in a patient with glaucoma and decreased central corneal thickness as well as findings consistent with lacrimo-auriculo-dento-digital syndrome (MIM 149730) . Peters anomaly in our patient and decreased corneal thickness associated with glaucoma in the previously reported patient may suggest that the Arg343 residue of TP63 plays a role in corneal development.
Some limitations of our study include the variety of ASD diagnoses among our patient sample. Our study encompasses patients with Peters anomaly, aniridia, AR syndrome, and PCG. Since the sample size is small for each ASD condition, it is difficult to assess the mutation detection rate in each ASD condition. Moreover, we did not identify a potentially causative variant in over half of our probands. With the available ES data and an increased cohort in the future, we expect to identify more variants to characterize the genetic features of ASD in South Florida. Finally, variants located in regulatory regions such as introns, promotors, and enhancers, may be studied with genome sequencing in the future.