Genes · 11 authors, 5 centres
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This study achieved a 100% diagnostic rate in seven consanguineous Pakistani families with anophthalmia/microphthalmia by combining targeted FOXE3 sequencing with genome sequencing. Pathogenic variants were identified in FOXE3, PXDN, and VSX2, including a novel deep intronic splice variant in PXDN confirmed by minigene assay. The findings highlight the clinical utility of genome sequencing for detecting non-coding variants and improving genetic diagnosis in severe bilateral A/M.
**Background:** Anophthalmia and microphthalmia (A/M) are severe congenital eye malformations with a combined prevalence of 1 in 10,000 births. Despite advances in genomic technologies, over 50% of A/M patients lack a molecular diagnosis. Genetic factors are the primary cause, with over 90 associated genes, but diagnostic rates remain low, especially for unilateral cases. This study aimed to evaluate a combined approach of targeted single-gene testing and genome sequencing to improve diagnostic yield in a consanguineous Pakistani cohort.
**Methods:** Seven consanguineous families (20 affected individuals) with bilateral A/M (10 microphthalmia, 10 anophthalmia) from Pakistan were enrolled. All families had an unknown molecular basis. Initial screening used Sanger sequencing of the FOXE3 coding region, given its high prevalence in similar cohorts. For FOXE3-negative probands, genome sequencing was performed on a BGISeq500 platform with 30-fold median coverage. Variant prioritization focused on rare (MAF ≤0.01) homozygous or compound heterozygous variants in 147 A/M-associated genes, using in silico tools (CADD, REVEL, SpliceAI). A novel deep intronic PXDN variant was functionally validated via in vitro minigene splice assay in HEK293T cells.
**Key Results:** All seven families received a genetic diagnosis (100% solve rate). Targeted FOXE3 sequencing identified two previously reported pathogenic variants in four families: a homozygous nonsense variant (c.720C>A; p.(Cys240*)) in families MA102, MA125, and MA203, and a homozygous missense variant (c.289A>G; p.(Ile97Val)) in family MA201. In the remaining three families, genome sequencing revealed: a known pathogenic PXDN frameshift variant (c.2568del; p.(Cys857Alafs*5)) in family MA193; a novel 13bp deletion in VSX2 (c.413_425del; p.(Ser138*)) in family MA174; and a novel deep intronic splice variant in PXDN (c.3609-1307G>A) in family MA144. The intronic variant was predicted by SpliceAI (acceptor gain score 0.97) to create a pseudoexon. The minigene assay confirmed activation of a 137bp pseudoexon (c.3609-1305_3609-1169) leading to an out-of-frame insertion and premature stop codon (p.Arg1203Serfs76*), with no wildtype transcript observed. All variants were classified as (likely) pathogenic per ACMG/AMP guidelines. Phenotypic heterogeneity was noted: e.g., the same FOXE3 nonsense variant caused anophthalmia in one family but microphthalmia with corneal opacity in others; the PXDN intronic variant showed variable severity within family MA144.
**Clinical Implications:** This study demonstrates that combining targeted FOXE3 sequencing with genome sequencing can achieve a 100% diagnostic rate in severe bilateral A/M families, offering a cost-effective strategy. The identification of a novel deep intronic PXDN variant underscores the importance of non-coding regions in A/M genetics and the value of genome sequencing over exome sequencing for detecting such variants. These findings improve genotype-phenotype correlations, enable better genetic counseling, and may guide patient management. The high prevalence of FOXE3 variants (57%) in this Pakistani cohort suggests that pre-screening this gene is cost-effective in similar populations. Limitations include the small sample size and lack of advanced ocular imaging (OCT, MRI) for complete phenotypic characterization.