A retrospective study, reviewing consecutive records of patients who completed genomic testing for cataracts through the North Thames Genomic Laboratory Hub over a 9-year period (January 2016–December 2025) was undertaken.

The study was registered in our institution as a service evaluation (registration number 4552).

Patients were excluded if they had not been clinically reviewed and phenotyped as children under the care of Great Ormond Street Hospital Ophthalmology or Clinical Genetics services. Informed consent was taken from the families presented as case studies.

The clinical health records of 119 probands with congenital or developmental cataracts, with or without wider congenital ocular anomalies or systemic features, were reviewed. Detailed information on family history, pregnancy, delivery, developmental, health, and ophthalmic findings was collated. All probands and relevant family members underwent comprehensive ophthalmic assessments including acuity assessment, orthoptic and ophthalmologic examination, and, in cases undergoing surgery, biometry, keratometry, and anterior segment imaging. Electrodiagnostic assessment was carried out when clinically indicated. Where appropriate, wider nonophthalmic clinical phenotyping of developmental and systemic features was completed by clinical genetics.

DNA was isolated from peripheral venous blood in all proband and relevant relatives. Panel (exome) analysis was performed on a singleton basis (with parental segregation following when indicated), whereas the majority (35 of 43) of WGS cases were analyzed as trios (3 as duos, 5 as singletons). Between January 2016 and November 2022, testing was exome based. Initially, this consisted of a clinical exome (GOSHome), a curated panel that evolved from the research developed the Oculome panel test. This transitioned to singleton whole exome sequencing with virtual panel application in 2020. From 2021, gene selection was in accordance with the NHS-wide Genomic Medicine Service test directory, using the R31 gene list from PanelApp.

Exome-based sequencing was completed on the Illumina HiSeq 2500 platform with the Agilent SureSelect Focused Exome +1 capture. Sequence data were generated across the full capture region of >5000 genes, with analysis limited to genes listed in the technical report; this involved selective analysis of between 91 and 109 genes associated with congenital and developmental cataracts. Known benign polymorphisms and sequence variants that were considered unlikely to be pathogenic were not reported.

Between December 2023 and December 2025, routine analysis transitioned to WGS. This NHS-wide centralized process was conducted by Illumina, with subsequent analysis of variants prioritized by the Genomics England bioinformatics pipeline for the GMS R31 gene panel. The analysis included interrogation of tier 1 (loss-of-function or de novo protein-altering variants in “green,” diagnostic grade, panel genes) and tier 2 (other variant types in panel genes). Tier 3 (other filtered variants) was restricted to relevant de novo variants (trio analysis only) and prioritized variants identified through phenotype-driven approaches, such as Exomiser ( https://www.sanger.ac.uk/tool/exomiser/ ). Again, known benign polymorphisms and sequence variants that were considered unlikely to be pathogenic were not reported.

Sequence variant classification was performed in accordance with the 5-tier American College of Medical Genetics and Genomics (ACMG) guidelines, that is, pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign.

Interpretation considered the evaluation of multiple evidence types, including population frequency, computational predictions, functional studies, segregation data, allelic data, and genotype-phenotype correlations. Copy number variant classification used similar guidance, including the most up-to-date joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen).

Among the 119 probands, the majority were sporadic cases, with only 12.6% (15 of 119) having a documented family history. Of the familial cases, most had a history suggestive of autosomal dominant inheritance. Consanguinity was reported in a small proportion. All but 3 individuals had bilateral cataracts at the time of genetic testing. The majority were diagnosed within the first 12 months of life with apparently isolated congenital cataracts, although many also showed minor additional ocular findings, either directly related to the cataract or subsequent treatment. These included refractive errors, strabismus, amblyopia, and glaucoma. A smaller number presented with microcornea, microphthalmia, or posterior lenticonus.

Patients with isolated cataracts and/or additional ocular features (52.1%, 62 of 119) were classified as having nonsyndromic cataracts (NSC). Those with extraocular manifestations—including neurodevelopmental disorders, impaired growth, neurologic abnormalities, systemic disease (eg, cardiac, renal, or skeletal differences), and/or distinctive facial features—were classified as having syndromic cataracts (SCs; 47.8%, 57 of 119).

A total of 50 diagnostic (pathogenic/likely pathogenic) variants in 32 genes were identified. Diagnostic rates were higher for familial (80.0%, 12 of 15) compared to sporadic cases (36.5%, 38 of 104), and in clinically nonsyndromic cataract (51.6%, 32 of 62) compared with syndromic cataract (SC) (31.6%, 18 of 57). The overall diagnostic yield was 42.0%. Autosomal dominant genes (either inherited or de novo ) were the most frequently reported (66%, 33 of 50), followed by autosomal recessive (20%, 10 of 50) and then X-linked (14%, 7 of 10).

The diagnostic yield for both NSC and SC was higher for those undergoing WGS (48.8%, 21 of 43) compared with exome sequencing (38.2%, 29 of 76) ( Figure 2 ). However, using Fisher exact test, there was no statistically significant difference in the diagnostic rate between the exome/panel and WGS groups ( P =.334). Although WGS showed a higher raw diagnostic yield, the difference was not statistically significant with this sample size (OR 1.54, 95% CI 0.68-3.52).

Comparison of diagnostic yield by sequence modality for nonsyndromic cataracts (NSCs) and syndromic cataracts (SCs).

Among the reported NSC variants, crystallin genes accounted for 24% (12 of 50) ( Figure 3 ), including CRYAA, CRYBA1, CRYBB2, CRYBB1, CRYBB3, CRYGD , and CRYGC. Connexin gene variants (gap junction proteins) GJA8 ( Figures 4 and 5 ) and GJA3 accounted for 12% (6 of 50), and transcription factors, including PAX6, HSF4 , and PITX3 , for 10% (5 of 50). Variants in extracellular matrix and cytoskeletal proteins were identified in 6% (3 of 50) and 4% (2 of 50), respectively. Nonstructural protein gene variants including FYCO1 ( Figure 6 ) and CPAMD8 accounted for 2% (5 of 50). The remaining 38% (19 of 50) of variants were reported in 14 genes associated with a wide range of syndromic disorders. Two unrelated families had identical variants in CTDP1 , and 5 families had different variants in NHS ( Figures 7 and 8 ).

Proportion of diagnostic gene variants by group and category.

A, B. GJA8 mutation with mild lens phenotype (Right eye 4A , Left eye 4B ).

A, B. GJA8 mutation with severe microphthalmia, lens opacification, and anterior dysgenesis phenotype (Right eye 5A , Left eye 5B ).

A, B. Homozygous FYCO1 mutation with bilateral dense lens opacities (Right eye 6A , Left eye 6B ).

A, B. NHS gene mutations causing irregular congenital nuclear cataracts (Right eye 7A , Left eye 7B ).

A, B. NHS gene mutations causing congenital nuclear cataracts (Right eye 8A , Left eye 8B ).

In those cases, with diagnostic variants identified, 64.0% (32 of 50) were in probands with NSC at the time of assessment. Of these, 84.3% (27 of 32) were in genes associated with isolated congenital cataracts, with or without other ocular anomalies. The remaining 15.6% (5 of 32) were in genes with a spectrum of disease that may include wider systemic features ( GALK1, NHS x 2, OPA3 , and a 2.3-Mb contiguous gene deletion including PAX6 at chromosome position 11p.13). The proband with biallelic variants in GALK1, c.766C>T p.(Arg256Trp) and the proband with a de novo heterozygous variant in OPA3, c.277G>A p.(Gly93Ser), presented with isolated cataracts between the ages of 5 and 6 years.

No other ocular or systemic concerns were noted on initial clinical assessment. The proband with a maternally inherited 11p13 deletion presented at birth with bilateral cataracts and subtle iris hypoplasia ( Figure 9 ). Two probands with pathogenic NHS variants presented with visually significant cataracts in the neonatal period, 1 male c.295del p.(Ile99SerfsTer97maternal) ( Figure 7 ) and 1 female (c.2536A>T p.(Lys846Ter de novo) ( Figure 8 ). Neither were found to have wider ocular or systemic features either at presentation or at review at 12 months.

A, B. PAX6 mutation with bilateral dense lens opacities, iris hypoplasia, and anterior segment dysgenesis (Right eye 9A , Left eye 9B ).

Thirty-six percent (18 of 50) of diagnostic variants were in probands clinically felt to have syndromic disorders. The mode of inheritance was autosomal dominant in 7 (parentally acquired or de novo ), autosomal recessive in 6, and X-linked in 5. Three variants were reported in genes well established to be associated with NSCs ( BPSP2 and 2 in GJA8 ), and in at least 1 proband, a second diagnosis was identified to explain the systemic features. The remaining 15 variants arose in 11 genes ( ALDH18A1, BCOR, COL2A1, COL4A1, CTDP1 [×2], DYNC1H1, DYRK1A, FTL, NHS [×3], OCRL , and RAB3GAP1 ) and all were novel, with the exception of the recurrent Roma founder mutation in CTDP1 (c.863+389C>T), identified in 2 apparently unrelated probands.

Congenital cataract was the presenting feature in the probands with variants in OCRL, BCOR , and NHS , whereas a broader constellation of features was noted in probands with variants in the remaining genes. The more common associated systemic findings were developmental delay (particularly low tone in infancy), intellectual disability, distinctive facial features, and growth concerns. The 3 male probands with variants in NHS (c.586dup p.(Glu196Glyfs*3), c.3659dup p.(Asn1220Lysfs*5), c.3871C>T p.(arg975*) had cataracts, mild developmental delay, and abnormal dentition (widely spaced, “screwdriver,” or unusually shaped teeth), consistent with a diagnosis of Nance-Horan syndrome.

Of the 12 heterozygous variants found in 7 crystallin genes, 5 were missense in Greek key motifs I, II, and IV ( CRYBB2 : c.446G>T p.(Gly149Val), CRYBB3 : c.466G>A p.(Gly156Arg), CRYGC : c.233C>T p.(Ser78Phe), CRYGC : c.173T>C p.(Leu58Pro), CRYGD : c.70C>A p.(Pro24Thr), 2 in the α-crystallin domain (identical variants in unrelated families, CRYAA : c.346C>T p.(Arg116Cys), and 1 in the N-terminal domain CRYAA : c.61C>T p.(Arg21Trp). All are locations where mutations are well established to disrupt protein folding, solubility, and chaperone function, leading to lens opacification and congenital cataract. The remaining 4 variants are expected to be loss of function: 1 a multiexon deletion of CRYBB1 ; 1 a nonsense mutation in the C-terminal Greek key motif IV CRYBA1 : c.(519T>G p.Tyr173*); and 2 intron variants predicted to lead to aberrant splicing ( CRYBA1 : c.32-3C>G, CRYBA1 : c.215+1G>A).

All but 2 ( CRYBA1 : c.32-3C>G, CRYBA1 : c.519T>G p.(Tyr173*) have been previously reported in the medical literature, although similar splice site and truncating mutations in CRYBA1 have been reported as disease-causing. ^,,, Two of the variants were inherited, and the remainder were de novo . All patients presented with bilateral congenital cataracts, and 7 exhibited additional features including nystagmus, strabismus, and raised intraocular pressure. The 2 probands with microphthalmia had variants in the same domain (Greek key motif IV) of different genes, CRYBB2 c.446G>T p.(Gly149Val) and CRYBB3 c.466G>A p.(Gly156Arg). ^,,,,,,,,,,

Variants in gap junction proteins accounted for 6 diagnoses (1x GJA3 and 5x GJA8 ), all were missense and half were novel. Four of the variants were located in the first extracellular loop ( GJA3 : c.199G>C p.(Asp67His); GJA8 : c.130G>A p.(Val44Met), c.136G>A p.(Gly46Arg), c.142G>A p.(Glu48Lys), 1 in the second transmembrane domain GJA8 : c.263C>A p.(Pro88Gln), and 1 in the N-terminal domain GJA8 : c.20T>C p.(Leu7Pro). All were predicted to disrupt gap junction trafficking and channel function and result in a spectrum of developmental eye disorders. ^,,,,,, Aside from congenital cataracts, 2 patients had wider eye manifestations, including nystagmus and esotropia ( GJA8 : c.142G>Ap.(Glu48Lys) and microphthalmia, microcornea and esotropia ( GJA8 : c.136G>A p(Gly46Arg) ( Figure 5 ). Additional systemic features were noted in one patient who had a second chromosomal diagnosis (22q11.2 microdeletion).

A total of 17 VUSs were reported in 14 genes ( Figure 10 ). Except for GJA8 (4 variants), all were single variants in individual genes. Nonsyndromic genes accounted for 47.1% (8 of 17) and syndromic genes 52.9% (9 of 17). All were rare variants with low frequency in population databases and predicted to be deleterious through in silico tools; however, further evidence of pathogenicity was lacking. Recognized incomplete penetrance and variable expressivity of many of these genes limited the application of the ACMG criteria PP4 (prospective diagnosis in keeping with clinical features).

Spectrum of VUSs (variants of unknown significance).

In addition, not having access to parental samples also prevented the application of de novo status to likely causative genes. Of the 4 VUSs in GJA8 (c.130G>C p.(Val44Leu), c.299T>A p.(Val100Asp), c.776C>T p.(Ser259Phe), and c.116C>G p.(Thr39Arg), only the latter, located in the first transmembrane domain, had been previously reported in association with congenital cataract and microphthalmia. The remaining variants were either located in extracellular loops critical for channel formation and function (p.(Val44Leu) and p.(Val100Asp)) and with known analogy to known pathogenic variants, or in the C-terminal domain with possible effects on channel regulation (p.(Ser259Phe)). ^,