Introduction

Structural eye anomalies, including anophthalmia, microphthalmia and coloboma, are genetically heterogeneous disorders, occurring as part of identifiable syndromes in 20–45% of cases [1]. Over 140 genes are currently tested on diagnostic panels, including the UK PanelApp ‘Structural Eye Disease’ (https://panelapp.genomicsengland.co.uk/panels/509/).

Mendelian Disorders of the Epigenetic Machinery (MDEMs) are an expanding group of conditions caused by variants in epigenetic regulators, including CHD7 (CHARGE syndrome, OMIM: 214800) and KMT2D (Kabuki syndrome 1, OMIM: 147920). Recently, monoallelic variants in KDM2B (Lysine demethylase 2B) were associated with a new MDEM presenting with developmental delay (DD), variable systemic features and a characteristic DNA methylation profile (‘KDM2B-associated episignature’) [2]. To date, three 12q24.31 deletions and 22 pathogenic/potentially pathogenic single-nucleotide variants (SNVs) have been reported in 28 families [2,3,4]. Of these, 17 different SNVs clustered within the small zinc-finger domain CxxC [5, 6]. Intriguingly, KDM2B-CxxC variants generated a specific ‘sub-episignature’, suggesting a distinct molecular effect [2]. Compared with individuals carrying non-CxxC variants, CxxC cases additionally presented with congenital heart defects (17/21) and kidney agenesis (6/21). Over half (13/21) had ophthalmological findings; while these mainly included strabismus, myopia and astigmatism, 3/13 individuals exhibited severe structural eye anomalies (not observed in non-CxxC cases), suggesting that KDM2B-CxxC variants influence eye morphogenesis.

To gain further insight into this, we analysed 706 families with developmental eye anomalies, identifying two new cases with pathogenic KDM2B-CxxC missense variants. Both displayed complex structural eye disorders and extra-ocular features consistent with those previously reported in KDM2B-CxxC cases.

Subjects and methods

Families 1 and 2 were identified through whole exome/genome sequencing (WES/WGS) analysis of 706 families with structural eye anomalies (456/706 genetically undiagnosed).

Family 1: WES of proband and maternal DNA was performed by Psomagen (Rockville, US-MD) [7] and SNVs and copy number variants (CNVs) analysed using VarSeq (Golden Helix, Bozeman, US-MT) [8].

Family 2: WGS of proband and parental DNA was performed by Theragen Bio (Republic of Korea). SNVs were analysed using an in-house pipeline prioritising rare coding and canonical splice variants. CNVs and structural variants were assessed using Optical Genome Mapping (Bionano Genomics, US-CA).

DNA methylation profiles were assessed using the EpiSign assay [9, 10]. KDM2B variants were validated using Sanger sequencing. Pathogenicity was ascribed using ACMG/AMP criteria following the UK-ACGS guidelines [11, 12].

Results

Family 1

Case 1 (Individual II.1, Fig. 1A) is a 9-year-old black male, born at full-term by normal delivery (birth weight: 3.15 kg). He was diagnosed with bilateral congenital glaucoma (buphthalmos). He underwent right trabeculotomy and trabeculectomy at 14 days of age and Ahmed glaucoma valve (AGV) insertion at 1 month of age (right) and 2 months of age (left). At 9 years of age he exhibited right exotropia, bilateral myopia with buphthalmos, bilateral iris hypoplasia with corectopia, left posterior embryotoxon, a small left anterior capsule cataract (detected at 8 months of age and possibly AGV-related) and a small left chorioretinal coloboma (Fig. 1B). He displayed facial asymmetry, high forehead, bitemporal narrowing, malar hypoplasia, small ears and short stature (8 years of age: 117.2 cm; just <3rd centile). He had generalized delayed dental development and required removal of two persistent primary teeth at 8 years of age; at 9 years of age, he had partially erupted molars and persistent primary incisors. He also had DD with possible autism spectrum disorder.

Fig. 1: Novel families with pathogenic KDM2B variants and structural eye anomalies.
Fig. 1: Novel families with pathogenic KDM2B variants and structural eye anomalies.
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A Pedigree of Family 1. The proband (II.1) carries the KDM2B variant c.1841G>C;p.(Arg614Pro), absent in the mother (I.2). Paternal DNA was not available (NA). B Ocular images of Proband II.1 (Family 1). Right eye (OD) shows an increased cup to disc ratio (0.80); left eye (OS) shows an increased cup to disc ratio (0.65) and coloboma inferior to the optic nerve (arrow). C Pedigree of Family 2. The proband (II.4) carries the de novo KDM2B variant c.1880G>C;p.(Cys627Ser). Her older sister is unaffected. Triangles indicate miscarriages (wk weeks of gestation). D Photographs of Proband II.4 (Family 2). Left panel: image of the proband as a baby showing bitemporal narrowing, straight eyebrows, underdeveloped left socket (left vestigial eye remnant), right microphthalmia, smooth philtrum, thin upper lip and fleshy lower lip. Right panel: images of the proband’s eyes at 18 years of age. Right eye (OD): mild microphthalmia with disorganised anterior segment; left eye (OS): anophthalmia.

Chromosomal microarray revealed a 1q21.3 deletion (chr1:152,028,001-152,250,046, hg38), not detected in maternal WES data. This was deemed non-contributory, since there are no haploinsufficient genes within the region. WES identified a heterozygous KDM2B missense variant [NM_032590.5:c.1841G>C;p.(Arg614Pro)], absent from gnomAD v4.1.0. The change was not detected in the unaffected mother; paternal DNA was unavailable. The variant, located within the CxxC domain, is predicted to be damaging/probably damaging by multiple in silico tools (dbNSFP v5.3, https://www.dbnsfp.org/). The same change is also reported in ClinVar (SCV005079302; phenotype unavailable) and in a published case [4]; an additional case displayed a different amino acid change at Arg614 [NM_032590.5:c.1841G>T;p.(Arg614Leu)] [3]. While previously published cases with Arg614 variants did not undergo DNA methylation testing, EpiSign analysis of Case 1 detected the KDM2B-associated episignature with ‘moderate’ confidence. The variant was classified as pathogenic (Table 1).

Table 1 Phenotypic and genetic findings of the two cases presented in this study.
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Following genetic diagnosis, renal ultrasound identified a solitary right kidney, while echocardiography was normal.

Family 2

Case 2 (Individual II.4, Fig. 1C) is a 19-year-old female, born at 41+ 6 weeks’ gestation by normal delivery after induction, following a pregnancy complicated by gestational diabetes. Her birth weight was 4.45 kg (90–97th centile), head circumference (HC) on the 50th centile. At one week of age, she was diagnosed with left anophthalmia (3.5 mm anophthalmic remnant) and mild right microphthalmia (16 mm axial length) (Fig. 1D). Visual evoked potentials were absent on the left and probably absent on the right. Sequential left socket expansion using hydrophilic expanders was commenced. At 5 months of age, MRI revealed delayed myelination and a 3 mm intradiploic epidermoid cyst midline in the frontal bone, while confirming a left anophthalmic remnant with reduced orbital size and right microphthalmia. Optic nerves and chiasm were not demonstrable; optic tracts were hypoplastic. She had mildly delayed motor development (she rolled over at 6–7 months of age, sat unsupported at 8–9 months of age, never crawled and walked at 17–21 months of age), although this progression was within the normal range for a child with visual impairment. She had no speech delay, cognitive impairment or behavioural issues. At 17 months of age, her HC had fallen to <2nd centile (length: 50th centile, weight: 9th centile). Echocardiography revealed an isolated ostium secundum atrial septal defect (ASD) surgically closed at 3 years 4 months. Teeth were generally crowded; both upper canines failed to erupt normally and had been removed. Other features included bitemporal narrowing and hypermobility. Renal ultrasound confirmed normal kidneys. At 18 years of age, she exhibited microcephaly (HC: 52.3 cm [0.4th–2nd centile], height: 160 cm [25th centile], weight: 48.5 kg [9th centile]), left anophthalmia, right microphthalmia with a disorganised anterior segment and a small iris coloboma/corectopia (Fig. 1D) and no light perception in either eye.

Case 2’s WGS identified a de novo KDM2B missense variant [NM_032590.5:c.1880G>C;p.(Cys627Ser)], absent from gnomAD v4.1.0. The change, predicted to be damaging by multiple in silico algorithms (dbNSFP v5.3), affects a CxxC residue directly involved in Zn2+ binding [2]. A different variant affecting Cys627 [NM_032590.5:c.1880G>A;p.(Cys627Tyr)] was previously reported as pathogenic [2, 3]. Case 2 tested positive for the KDM2B-associated episignature with ‘strong’ confidence (EpiSign v5). The variant was classified as pathogenic (Table 1). No other SNVs/CNVs of relevance to her eye phenotype were identified.

Discussion

We describe two individuals with KDM2B variants [p.(Arg614Pro) and p.(Cys627Ser)] manifesting significant developmental eye anomalies and variable neurodevelopmental, renal and cardiac phenotypes.

KDM2B has two main isoforms, both containing a CxxC domain, a plant homeodomain (PHD), an F-box and a Leucine-rich repeat (LRR). The full-length isoform additionally includes a JmjC histone demethylase domain (Fig. 2). KDM2B binds promoter-associated unmethylated CpG islands via CxxC [6, 13], protecting them from de novo methylation [14, 15]. Moreover, in mouse embryonic stem cells (mESCs), Kdm2b recruits the non-canonical Polycomb Repressive Complex 1.1 to early lineage gene promoters, helping establish epigenetic transcriptional programmes essential for development [6,13].

Fig. 2: Schematic of KDM2B with pathogenic variants reported in this and previous studies.
Fig. 2: Schematic of KDM2B with pathogenic variants reported in this and previous studies.
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Protein domains are reported according to UniProt entry Q8NHM5. Both the short (SF) and the full-length (FL) isoforms contain a DNA-binding domain (CxxC), a plant homeodomain (PHD), an F-box and a Leucine-rich repeat (LRR). The amino acids forming the CxxC zinc-finger domain are indicated with the single-letter amino acid code in the bottom panel. The CxxC domain contains two cysteine-rich clusters (CxxCxxCx4CGxCxxC and CxxRxC), with eight cysteines (indicated in red) coordinating two Zn2+ ions in a tetrahedral manner and a linker region containing the highly conserved motif KFGG (Lys-Phe-Gly-Gly) and the DNA-binding motif KQ (Lys-Gln) [5]. The latter, together with the upstream residue (Met), directly interacts with the CpG dinucleotide. The two variants identified in this study are indicated in bold, together with pathogenic variants from previous studies [2,3,4]. Family identifiers (square brackets) are reported according to the numbers used in previous publications: van Jaarsveld et al. [2] in purple, van Oirsouw et al. [3] in orange, Gomes et al. [4] in green, this study in black. Red dots indicate variants where at least one case exhibited the KDM2B episignature; black dots indicate variants for which methylation analyses were not performed.

KDM2B variants have recently been implicated in a new MDEM, with CxxC emerging as a hotspot domain [2,3,4]. In addition to speech delay (21/21), motor delay (18/21), intellectual disability (ID) or learning difficulties (10/13), behavioural issues (9/19), microcephaly (4/18) or macrocephaly (2/18), brain (7/12) and skeletal anomalies (14/21), CxxC cases also exhibited congenital heart (17/21), kidney (6/21) and structural eye anomalies (3/21) (Supplementary Table).

By screening families recruited for structural eye anomalies, we identified two new cases with KDM2B-CxxC variants. Case 1 [p.(Arg614Pro)] displayed bilateral iris hypoplasia with corectopia, left posterior embryotoxon and chorioretinal coloboma, left kidney agenesis, facial asymmetry, small ears, short stature, DD and possible autism. A 2-year-old boy with the same variant [p.(Arg614Pro) [4]] showed solitary kidney, cardiac anomalies, myopic astigmatism, plagiocephaly, facial asymmetry and global DD. A 15-year-old boy carrying the variant p.(Arg614Leu) [3] exhibited simple ears, short stature, speech and motor delay, severe ID, behavioural disorders, but no eye anomalies. Case 2 [p.(Cys627Ser)] had left anophthalmia, right microphthalmia with anterior chamber anomalies, apparent optic nerve aplasia, microcephaly, ASD, but no kidney anomalies. A previously described individual carrying the variant p.(Cys627Tyr) [2] had cardiac defects, kidney agenesis, short stature, mild DD, behavioural issues and normal eyes, suggesting that congenital anomalies also occur variably with Cys627 variants. Importantly, Case 2 had mild early motor delay attributable to her visual impairment, but no speech, cognitive or behavioural issues. Both Case 1 and 2 had anomalous dental development (persistent primary dentition and/or delayed tooth eruption), also described in another CxxC case [p.(Arg649Pro) [3]], possibly expanding the KDM2B spectrum. Interestingly, dental, cardiac and structural eye anomalies also occur in individuals with variants in BCOR (OMIM: 300485) [16], a PRC1.1 component interacting with KDM2B.

Cases 1 and 2 tested positive for the KDM2B-associated episignature, supporting the deleterious role of these variants. Case 1’s episignature was called with moderate confidence, potentially reflecting a hypomorphic effect of p.(Arg614Pro) [10]. Unlike Cys627, Arg614 is not directly involved in Zinc binding; Arg614 variants were predicted to affect local protein structure instead [3]. However, as seen for other recurrent KDM2B-CxxC variants, Case 1 and the previously described individual with p.(Arg614Pro) displayed different heart and eye phenotypes, suggesting that modifying factors (genetic, environmental and/or stochastic) also contribute to the variable expressivity of congenital anomalies. Given KDM2B’s widespread role in gene repression [17], variants in target genes or other developmental regulators of gene expression may also influence the phenotypic outcome.

Mouse models support the importance of Kdm2b in the heart, eye and neuro-development. Homozygous deletions disrupting both Kdm2b isoforms are embryonically lethal, causing severe malformations, including neural tube, craniofacial and cardiovascular defects [14, 18]. Deletion of the Kdm2b-CxxC domain (Kdm2bΔCxxC) also led to semi-lethality and congenital anomalies in heterozygosity [19]. While ocular phenotypes were not assessed in Kdm2bΔCxxC animals, ~40% of mouse embryos lacking full-length Kdm2b displayed neural tube defects, retinal coloboma and expanded neuroretina, highlighting Kdm2b’s involvement in eye development [20].

In summary, our study supports the role of KDM2B-CxxC dysfunction in heart, kidney and eye malformations and emphasises the importance of testing KDM2B and its episignature in individuals with structural eye anomalies, particularly when renal and/or cardiac findings are present.

Web resources

ClinVar: https://www.ncbi.nlm.nih.gov/clinvar/. dbNSFP: https://www.dbnsfp.org/. gnomAD: https://gnomad.broadinstitute.org/. OMIM: https://www.omim.org/. PanelApp: https://panelapp.genomicsengland.co.uk/panels/509/. UniProt: https://www.uniprot.org/.