Genetic and environmental factors contributing to anophthalmia and microphthalmia: Current understanding and future directions

Abstract

Anophthalmia is defined as a complete absence of one eye or both the eyes, while microphthalmia represents the presence of a small eye within the orbit. The estimated birth prevalence for anophthalmia is approximately 3 per 100000 live births, and for microphthalmia, it is around 14 per 100000 live births. However, combined evidence suggests that the prevalence of these malformations could be as high as 30 per 100000 individuals. Microphthalmia is reported to occur in 3.2% to 11.2% of blind children. Anophthalmia and microphthalmia (A/M) are part of a phenotypic spectrum alongside ocular coloboma, hypothesized to share a common genetic basis. Both A/M can occur in isolation or as part of a syndrome. Their complex etiology involves chromosomal aberrations, monogenic inheritance pattern, and the contribution of environmental factors such as gestational-acquired infections, maternal vitamin A deficiency (VAD), exposure to X-rays, solvent misuse, and thalidomide exposure. A/M exhibit significant clinical and genetic heterogeneity with over 90 genes identified so far. Familial cases of A/M have a complex genetic basis, including all Mendelian modes of inheritance, i.e., autosomal dominant, recessive, and X-linked. Most cases arise sporadically due to de novo mutations. Examining gene expression during eye development and the effects of various environmental variables will help us better understand the phenotypic heterogeneity found in A/M, leading to more effective diagnosis and management strategies. The present review focuses on key genetic factors, developmental abnormalities, and environmental modifiers linked with A/M. It also emphasizes at potential research areas including multiomic methods and disease modeling with induced pluripotent stem cell technologies, which aim to create innovative treatment options.

Key Words: Anophthalmia; Microphthalmia; Eye development; SRY-Box 2; Orthodenticle homeobox 2

Core Tip: Anophthalmia (absence of one or both eyes) and microphthalmia (small eye within the orbit) are rare congenital conditions, affecting up to 30 per 100000 individuals. These malformations can occur independently or as a part of a syndrome, with complex genetic basis involving over 90 genes. Environmental factors like maternal infections, vitamin A deficiency, and toxin exposure also contribute to their development. Both conditions exhibit significant variability, making diagnosis and management challenging. This review discusses genetic and environmental influences on anophthalmia and microphthalmia, highlighting promising research areas such as multiomic approaches and stem-cell based models for potential therapeutic advancements.

INTRODUCTION

Anophthalmia and microphthalmia (A/M) are rare congenital eye disorders that present significant clinical challenges due to their impact on vision and overall quality of life[1]. Anophthalmia is defined as the complete absence of the eye globe within the orbit, with no observable ocular structures’ during a macroscopic clinical examination. Microphthalmia refers to an eye that is significantly smaller and underdeveloped, characterized by an axial length of less than 19 mm in children under one year of age or less than 21 mm in adults, as measured by B-scan ultrasound. These measurements are at least two standard deviations below the mean for the individual's age[2]. These conditions can occur in isolation or as part of a broader spectrum of ocular anomalies such as anterior segment dysgenesis, ocular coloboma, cataract, vitreoretinal dysplasia and can be unilateral or bilateral[3]. The clinical presentation of A/M can range from mild cases, where the eye is small but functional, to severe cases, with no visible ocular tissue, often accompanied by other craniofacial or systemic abnormalities[4].

The global incidence of A/M is estimated to be approximately 30 per 100000 individuals[1], though the prevalence can vary based on geographic, ethnic, and environmental factors[3]. In 32%-93% of A/M cases, other organ systems are affected by additional anomalies[3,5-8]. Furthermore, 20% of children with A/M and coloboma show delayed psychomotor development[9]. Despite its rarity, A/M accounts for a significant proportion of congenital blindness and visual impairment in children, particularly in underprivileged regions where early diagnosis and interventions are often challenging[1]. The burden of A/M extends beyond the visual impairment itself, often necessitating multidisciplinary management involving ophthalmologists, geneticists, pediatricians, and other specialists to address associated anomalies and provide comprehensive care[9].

The etiology of A/M is complex and multifactorial, involving a combination of genetic and environmental factors[10]. Genetic causes of A/M include mutations in key developmental genes, chromosomal abnormalities, and syndromic associations[10]. Environmental factors, such as maternal infections, exposure to teratogens, and nutritional deficiencies, have also been implicated in the development of these conditions[1]. The interplay between genetic predisposition and environmental exposures further complicates the understanding of A/M, as similar phenotypes can result from diverse etiological pathways[11].

Advances in molecular genetics and genomics have significantly enhanced our understanding of the genetic underpinnings of A/M[12]. Next-generation sequencing technologies have facilitated the identification of novel genetic mutations, providing insights into the complex gene networks involved in ocular development[4]. These findings have a substantial influence on the diagnosis, counseling, and management of patients and their families with A/M[6]. Concurrently, research into environmental risk factors underlines the role of maternal health and harmful exposures during pregnancy (especially in first trimester) in the development of these diseases[11].

Despite these advances, many questions remain unanswered regarding the defined mechanisms by which genetic and environmental factors contribute to the development of A/M[12]. The heterogeneity of these conditions, both in terms of clinical presentation and underlying causes, poses significant challenges for research and clinical practice[12]. Moreover, the interactions between genetic predispositions and environmental influences are complex and not yet fully understood, making it difficult to predict outcomes or develop targeted prevention strategies[10].

This review aims to provide a comprehensive overview of the current understanding of the genetic and environmental factors contributing to A/M. The present review explores in detail the genetic mutations and chromosomal abnormalities associated with A/M, as well as the environmental factors that increase the risk of these disorders. It also pinpoints the interplay of genetic and environmental variables, emphasizing epigenetic alterations and gene-environment interactions[11]. Finally, recent advances in research, including developments in genetic screening, animal models, and emerging therapeutic approaches, will be discussed along with future directions for research and clinical practice in the management of A/M[6].

PATHOPHYSIOLOGY OF A/M

The pathophysiological mechanisms underlying A/M are complex and involve disruptions in multiple genetic and developmental pathways critical for eye formation (Figure 1).

Figure 1 Key mechanisms for anophthalmia and microphthalmia. LncRNAs: Long non-coding RNAs.

Disruptions in early eye development

Human eye development begins around the third week of gestation in the anterior neural plate. Orthodenticle homeobox 2 (OTX2) is crucial for specifying the forebrain and enabling eye development by regulating key genes such as sine oculis homeobox homolog 3 (SIX3), retina and anterior neural fold homeobox gene (RAX), and paired box gene 6 (PAX6)[13-17]. OTX2 and SRY-Box 2 (SOX2) work together to regulate RAX, while SIX3 inhibits WNT signaling, activating critical eye field transcription factors (EFTFs) like PAX6 and LIM homeobox 2 (LHX2). This network of EFTFs governs eye development by decreasing inhibitory signals and activating genes such as bone morphogenetic protein 4 (BMP4), which are essential for eye formation[13,16,18-20]. Studies in various animal models[13,14,16,18] such as mouse, zebrafish, and xenopus have emphasized the essential roles of these genes in initiating eye formation[21-23].

Impaired formation of the lens placode

Between days 22 and 27 of gestation, SIX3 activates PAX6 and SOX2 in the surface ectoderm, crucial for forming the pre-placodal region[21,24-26]. Mice with conditional SIX3 deletion show reduced PAX6 and SOX2 levels and impaired pre-placodal thickening[4]. By days 27-28, LHX2 regulates BMP4 expression in the optic vesicle (OV)[14,18,21,24], inducing lens placode thickening[26-28]. BMP4-deficient mice fail to develop lenses, but this can be restored with exogenous BMP4, highlighting the critical function of LHX2 in regulating BMP4 and lens development[26,29].

Impaired optic cup morphogenesis

Between days 31-33 of gestation[18,21], the lens placode releases BMP and retinoic acid, inducing invagination of the OV[30-34]. Retinoic acid from the surrounding mesenchyme further supports this process[31,34]. In mice[18,21], a lack of retinoic acid due to retinaldehyde dehydrogenase knockouts results in incomplete OV invagination, which was rescued with maternal retinoic acid[30-34]. Studies performed in mouse models reported that, filopodia from the lens ectoderm are critical for coordinating lens and OV invagination, therefore, disruption of these filopodia inhibits lens pit development[21,35].

Impaired neural crest cell migration

Neural crest cells contribute to various eye structures, including the cornea, iris, and parts of the retina[36]. Impaired migration or differentiation of these cells due to mutations in genes such as PAX6, SOX10, OTX2, RAX, forkhead box (FOX) C1, BMP4, and FOXE3 can lead to malformations, resulting in microphthalmia or anophthalmia[37,38]. Additionally, conditional knockouts of Foxc2 in neural crest cells of mice exhibit microphthalmia, irregularly shaped irises, corneal vascularization and opacity[39]. Conditional knockout mice with LIM homeobox transcription factor 1-beta deletion (Lmx1b) specifically in neural crest cells, have a spectrum of anterior segment anomalies, such as microphthalmia, hypoplastic irises, reduced anterior chamber volume, aberrant corneal vascularization, and a marked reduction in the expression of the corneal endothelium marker i.e., neural cell adhesion molecule[40].

Disruptions in retinal development

Between 5-8 weeks of gestation, the OV invaginates to create the optic cup, resulting in retinal development. Disruptions/mutations in genes[41-45] such as SOX2, PAX6, OTX2, visual system homeobox 2 (VSX2) (also known as CEH10 homeodomain-containing homolog), RAX, cone rod homeobox (CRX), FOXE3, sonic hedgehog (SHH), growth/differentiation factor-6 (GDF6), BMP4, stimulated by retinoic acid 6 (STRA6), microphthalmia associated transcription factor (MITF), lysine-specific methyltransferase 2D (KMT2D), T-box transcription factor 3 (TBX3)[46-50] that regulate retinal cell proliferation and differentiation can cause anophthalmia or microphthalmia[51-53].

Apoptosis and proliferation imbalance

Aberrant regulation of cell proliferation and apoptosis during eye development can result in reduced eye size or the absence of eye tissue. Genes involved in controlling these processes, such as apoptotic protease activating factor-1 (APAF1), influence the balance between cell survival and death, and their dysfunction can lead to underdevelopment of the eye[54]. In mouse models of microphthalmia, an muscle segment homeobox gene 2 (Msx2) variant led to increased apoptosis by activating the caspase-3/8 pathway, while the loss of receptor-binding motif (Rbm) removed baculoviral inhibitor of apoptosis protein repeat-containing 2's (Birc2’s) inhibition of anti-apoptotic pathways[55]. Furthermore, in vitro studies using microphthalmia patient-derived models have shown upregulation of certain pro-apoptotic and cell cycle regulatory genes, reducing OV size[56,57].

In addition to intrinsic molecular signals guiding eye development, external stimuli shape the biophysical environment needed for optic cup formation and provide extracellular signals that create a supportive microenvironment for the developing eye[58-60]. The extracellular matrix (ECM), secreted by both the surface ectoderm and neuroectodermal OV, facilitates contact between these tissues, playing a pivotal role in regulating lateral OV growth and optic cup invagination[61,62]. The ECM also influences retinal progenitor proliferation and differentiation by affecting cell cycle dynamics[60].

Disruptions to laminin subunits α, β, and γ lead to ocular malformations, impaired basement membrane integrity, cell polarity, or focal adhesion assembly[63,64]. In zebrafish, disruption of the ECM protein nidogen (also known as entactin) hinders optic cup formation, although adding exogenous nidogen partially rescued optic cup invagination and fissure closure in double-mutant zebrafish lacking neural crest cells[58,65]. The ECM protein lumican (LUM) also controls axial length, as demonstrated in Lum^−/−/Fmod^−/− knockout mice and lum morpholino zebrafish, both showing elongated axial lengths[66,67]. Additionally, collagen type IV variants have been linked to ocular maldevelopment, with collagen alpha chain 1/4 (COL4A1/4) regulating retinal pigment epithelium (RPE) growth and function[68,69]. Despite the essential role of the ECM in ocular development, its dysfunction remains unexplored in the context of microphthalmia.

GENETIC MUTATIONS AND SYNDROMIC ASSOCIATIONS

A/M have a complex genetic background that includes Mendelian modes of inheritance i.e., autosomal dominant, autosomal recessive, and X-linked inheritance patterns, as well as rare de novo mutations. Non-penetrance and variable expressivity are commonly observed, suggesting the influence of genetic modifiers and environmental factors. Some A/M cases result from parental germline mosaicism, where mutations affect gonadal tissue but not ocular tissue. Chromosomal abnormalities, including aneuploidy, translocations, deletions, and duplications, account for 20%-30% of AM cases (Table 1)[1,70]. More than 90 genes linked with A/M (Tables 2 and 3, respectively) have been reported, majorly involved in eye development and the retinoic acid signaling pathway. Pathogenic mutations mainly include missense, nonsense, and splice-site variations.

Table 1 Common chromosomal abnormalities associated with anophthalmia and microphthalmia.

Type of chromosomal abnormality	Chromosome abnormality	Other features
Aneuploidy	Trisomy 9 mosaicism	Joint contractures, congenital heart defects, prenatal growth deficiency, learning difficulties, micrognathia, kyphoscoliosis
	Trisomy 13 (patau syndrome)	Holoprosencephaly, moderate microcephaly, coloboma, retinal dysplasia, cyclopia, cleft lip/palate, cardiac defects, genital abnormalities, 86% die within one year
	Trisomy 18 (edwards syndrome)	Polyhydramnios, single umbilical artery, small placenta, low fetal activity, learning difficulties, hypertonicity, hypoplasia of skeletal muscle, subcutaneous, adipose tissue, prominent occiput, low-set malformed auricles, micrognathia, cardiac defects
Triploidy	Triploidy	Large placenta with hydatidiform changes, growth deficiency, syndactyly, congenital heart defects, brain anomalies/holoprosencephaly
Deletion	4p (wolf-hirschhorn syndrome)	Growth deficiency, microcephaly, ocular hypertelorism, cranial asymmetry, learning difficulties, epilepsy, cleft lip/palate, anterior segment dysgenesis
	Deletion 7p15.1-p21.1	Cryptophthalmos, cleft lip/palate, choanal atresia
	13q-, ring 13	Microcephaly, learning difficulties, bilateral retinoblastoma, cardiac defects, hypospadias, cryptorchidism
	Deletion 14q22.1-q23.2	Pituitary hypoplasia
	18q	Midface hypoplasia, small stature, learning difficulties, hypotonia, nystagmus, conductive deafness, microcephaly, midface hypoplasia, genital abnormalities
Duplication	3q syndrome (3q21-ter dup)	Learning difficulties, growth deficiency, hypertrichosis, craniosynostosis, cardiac defects, chest deformities, genital abnormalities, umbilical hernia
	4p syndrome	Learning difficulties, epilepsy, growth deficiency, obesity, microcephaly, characteristic faces, genital abnormalities, kyphoscoliosis
	10q syndrome	Ptosis, short palpebral fissures, camptodactyly, learning difficulties, prenatal growth deficiency, microcephaly, heart and kidney malformations

Table 2 Genes associated with Anophthalmia (syndromic/non-syndromic) and its associated anomalies (https://hgmd.cf.ac.uk/ac/index.php).

Phenotype	Gene
Anophthalmia	ALDH1A3, MFRP, SOX2, RAX, OTX2, FRAS1, BMP4
Unilateral anophthalmia, isolated	ARG1
Anophthalmia and kidney abnormalities	BCOR
Anophthalmia and blepharophimosis	BMP4
A/M	CHD7, OTX2, STRA6, PUF60, SIX6
A/M with developmental delay	COL4A1
Developmental delay with anophthalmia	EPHA6
Anophthalmia and skeletal anomalies	GDF3
Anophthalmia, bilateral	GDF6, VSX2, OTX2
Holoprosencephaly with anophthalmia, branchial arch anomalies and central nervous system anomalies	GLI2
Bilateral anophthalmia, intellectual disability and rhizomelic skeletal dysplasia	MAB21L2
Waardenburg anophthalmia syndrome 4	MITF
Anophthalmia and isolated growth hormone deficiency	OTX2
Anophthalmia with corpus callosum hypoplasia, pituitary gland hypoplasia and vermian hypoplasia
Anophthalmia, growth delays, intellectual disability, and autism
Anophthalmia, hearing impairment, dysmorphic facial features and abnormal pituitary development causing growth failure
Anophthalmia, pituitary hypoplasia and ear anomalies
Bilateral anophthalmia, syndromic
Hypopituitarism with anophthalmia, dysmorphic facies, developmental delay and patent ductus arteriosus
Bilateral microphthalmia, anophthalmia or coloboma	PAX6
Non-syndromic A/M	PORCN
Brain malformations, anophthalmia, hepatomegaly, bile duct atresia and Müllerian duct agenesis	ptk7ie (isoform of PTK7)
Anophthalmia and sclerocornea	RAX
Anophthalmia, hypopituitarism, diabetes insipidus, bilateral cleft lip and palate
Microphthalmia, anophthalmia and coloboma disease	RERE, CDON, IQGAP1, CASK, MYO10, TENM3, POLR2A, WNT2B, KIF26B, MICU1, RBP4, FIG4, GDF6
Waardenburg anophthalmia syndrome	SMOC1
A/M, developmental delay, short stature and ataxic gait	SOX2
A/M, mental retardation, short stature and ataxic gait
A/M, mental retardation, short stature and unsteady gait
Anophthalmia and facial dysmorphism
Anophthalmia syndrome and dental anomalies
Anophthalmia with status dystonicus	SOX2
Anophthalmia-esophageal-genital syndrome
Anophthalmia, facial dysmorphism, delayed psychomotor development and language acquisition
Anophthalmia, global developmental delay, hypotonia, delayed language development and delayed gross motor development
Anophthalmia, hearing loss and brain abnormalities
Anophthalmia, hypogonadotropic hypogonadism and growth hormone deficiency
Bilateral anophthalmia and sensorineural hearing loss
Sox2 anophthalmia syndrome
Bilateral anophthalmia	SOX2, GJA8
Microphthalmia, anophthalmia and coloboma	SOX2, PAX6
Anophthalmia and diaphragmatic defects	STRA6
Anophthalmia syndrome
Colobomatous microanophthalmia	STRA6, TENM3
Anophthalmia and microphthalmia	VSX2, ALDH1A3, PXDN, FOXE3
A/M	VSX2, SOX2, RAX, FOXE3, ALDH1A3
Unilateral anophthalmia with accompanying malformations	WASHC5
Pulmonary hypoplasia, diaphragmatic anomalies, A/M and cardiac defects syndrome	WNT7B

A/M: Anophthalmia and microphthalmia; ALDH1A3: Aldehyde dehydrogenase 1 family member A3; ARG1: Arginase 1; BCOR: BCL6 corepressor; BMP4: Bone morphogenetic protein 4; CASK: Calcium/calmodulin dependent serine protein kinase; CDON: Cell adhesion molecule-related/downregulated by oncogenes; CHD7: Chromodomain helicase DNA binding protein 7; COL4A1: Collagen type IV alpha 1; EPHA6: Ephrin receptor ephA6; FIG4: FIG4 phosphoinositide 5-phosphatase; FOXE3: Forkhead box E3; FRAS1: Fraser extracellular matrix complex subunit 1; GDF3: Growth/differentiation factor 3; GDF6: Growth/differentiation factor 6; GJA8: Gap junction protein alpha 8; GLI2: GLI-kruppel family member 2; IQGAP1: IQ motif containing GTPase activating protein 1; KIF26B: Kinesin family member 26B; MAB21L2: Mab-21 like 2; MICU1: Mitochondrial calcium uptake protein 1; MITF: Microphthalmia associated transcription factor; MFRP: Membrane-type frizzled-related protein; MYO10: Myosin X; OTX2: Orthodenticle homeobox 2; PAX6: Paired box gene 6; POLR2A: Polymerase II, RNA, subunit A; PORCN: Porcupine O-acyltransferase; PTK7: Protein tyrosine kinase 7; PUF60: Poly(U) binding splicing factor 60-KD; PXDN: Peroxidasin; RAX: Retina and anterior neural fold homeobox; RBP4: Retinol binding protein 4; RERE: Arginine-glutamic acid dipeptide repeats; SIX6: SIX homeobox 6; SMOC1: SPARC related modular calcium binding 1; SOX2: SRY-box 2; STRA6: Stimulated by retinoic acid 6; TENM3: Teneurin transmembrane protein 3; VSX2: Visual system homeobox 2; WASHC5: WASH complex subunit5;WNT2B: Wnt family member 2B; WNT7B: Wnt family member 7B.

Table 3 Genes linked with microphthalmia (syndromic/non-syndromic) and its associated anomalies (https://hgmd.cf.ac.uk/ac/index.php).

Phenotype	Gene
Microphthalmia, isolated with coloboma 7	ABCB6
Microphthalmia and cataract	ALDH1A3, SHH, RAB3GAP2, FOXC1
Microphthalmia with facial clefting	ALX1
Microphthalmia, renal aplasia, hearing impairment, developmental delay, micropenis and cryptorchidism	ANOS1
Microphthalmia with linear skin defects	ARHGAP6
Craniosynostosis-microphthalmia syndrome	BCOR
Intellectual disability, cortical atrophy, microphthalmia and autism spectrum disorder
Lenz microphthalmia
Microphthalmia and cataracts
Microphthalmia with associated anomalies
Microphthalmia, syndromic 2
Pediatric total cataract with microphthalmia and microcornea
Anophthalmia, microphthalmia with sclerocornea and hydrocephalus	BMP4
Bilateral microphthalmia and unilateral cataract
Colobomatous microphthalmia	BRPF1, PUF60, TGFB2
Microphthalmia, syndromic	C12orf57, RAB3GAP2, TBC1D32, SCLT1, BCOR, KMT2D, NAA10, LAMB2, SLC18A2
Microphthalmia, colobomatous	C12orf57, SHH
Bilateral microphthalmia with accompanying malformations	CACNA1F, OSGEPL1, COL4A1, GAS2L2, IGFALS, LRP11, MAN1A1, CBR4, PKHD1L1, PRKG2, PDE6B, SUMF1, TBCE
Microphthalmia and/or coloboma	CAPN15
A/M	CHD7, OTX2, STRA6, PUF60, SIX6
Unilateral microphthalmia, isolated	CHD7, wnk4tv2 (isoform of WNK4), abcb9tv2 (isoform of ABCB9), wnk1tv2 (isoform of WNK1), FERMT1, CD22, MYO9B, ccn4tv3, EPB41, INTS11, IARS2RC3H1, ccn4tv3 (isoform of CCN4), EPB41, INTS11, IARS2RC3H1, AGRN, KRT31, PSME3IP, TUBGCP3, XKR4, MYCBP2, ABCC9, RPGRIP1L, MYH11, BHLHE22, ST18, FGF20, GRM8, ABCA13, CAPN11, NHLRC1, GRM6, MEGF10, PALLD, KMT2A, TYR, MFSD13A, HECTD2, MSRB2, ABCA1, KIFC2, FMNL2, ZP4, FRAS1, LIPH, MCCC1, P2RY14, CACNA2D3, SCN11A, GMPPA, PRKAG3, AOX1
Intellectual disability with microphthalmia	CNKSR1
A/M with developmental delay	COL4A1
Cataracts and microphthalmia
Microphthalmia with bilateral microcornea and Peters anomaly
Microphthalmia with linear skin lesions	COX7B, HCCS, NDUFB11
Pediatric perinuclear cataract with microphthalmia	CRYAA
Pediatric total cataract with microphthalmia	CRYBA4
Cataract and microphthalmia	CRYBB1
Cataract and microphthalmia	CRYBB3, BBS2, CRYAA
Nuclear cataracts with microphthalmia	CRYGC
Microphthalmia, fetal vasculature and vitreoretinal dysplasia	CTNNB1
Microphthalmia and agenesis of corpus callosum	DIS3L2
Mandibulofacial dysostosis, type Guion-Almeida, with microphthalmia, coloboma and retinal dystrophy	EFTUD2
Microphthalmia with iris hypoplasia	EPHA2
Colobomatous microphthalmia, ptosis, syndactyly and nephropathy	FAT1
Microphthalmia, coloboma, cardiac anomalies, renal dysfunction and limb malformations
Microphthalmia, and intellectual developmental disorder with dysmorphic facies and behavioral abnormalities	FBXO11
Microphthalmia and choroidal osteoma	FNBP4
Glaucoma, microphthalmia, iris anomalies, dysmorphic facial features, club foot, cognitive impairment and deafness	FOXC1
Bilateral microphthalmia and anterior segment dysgenesis	FOXE3
Microphthalmia with sclerocornea
Microphthalmia, nonsyndromic bilateral
Sclerocornea-microphthalmia-aphakia complex
Microphthalmia and anterior segment developmental anomalies	GDF6
Microphthalmia, congenital cataracts and microcornea	GJA3
Congenital cataract with microcornea, microphthalmia and posterior capsule defect	GJA8
Dystonia, bilateral ptosis, microphthalmia and speech and motor developmental delay	GNAL
Microphthalmia with linear skin lesions and autism spectrum disorder	HCCS
Microphthalmia, syndromic 7
Bilateral microphthalmia, isolated	hlcstv 4 (isoform of HLCS), ATRN, pax6tv32 (isoform of PAX6), TAS1R3, CERS2, NUMA1, TSFM, SLC5A11, PRKAG2, CFTR, GCK, THSD7A, CUZD1, VTI1A, UNC13D, CACNA1G, GFAP, LRP5
Colobomatous microphthalmia syndrome, X-linked	HMGB3
Ocular coloboma, microphthalmia and cataract	IPO13
Microphthalmia and coloboma	KIF17, MAB21L1
Microcephaly, long eyelashes, microphthalmia	KMT2A
Microphthalmia, failure to thrive, skeletal anomalies and neurological disorders
Unilateral microphthalmia with accompanying malformations	KRT23, VPS13D, MYH2, TRIM2, USP38, MYBPC3, KIAA2026, RYR2, MAP3K21, UNC13D, CHD7
Microphthalmia and aniridia	MAB21L1
Microphthalmia	MAB21L1, OTX2, MAB21L2, PRSS56, ALDH1A3, RAX, VSX1, PIEZO2, STRA6, SOX2, CHD7, RARB, DSC3, MFRP, RAB3GAP1, PXDN, GJA8, HMX1, MYO10, OLFM2, EPHA2, BCOR, TENM3, FOXE3, TMX3, VSX2, otx2tv5, CRYBA4, PAX6, VAX1, C12orf57, SEMA3E, RBP4, DHX38, ZNF219, GDF3, SIX6
Bilateral colobomatous microphthalmia, autosomal-dominant	MAB21L2
Microphthalmia/coloboma and skeletal dysplasia syndrome
Syndromic microphthalmia, type 14
Cataract, anterior segment dysgenesis and microphthalmia	MAF
Posterior microphthalmia, non-pigmented retinitis pigmentosa, optic nerve drusen, and retinoschisis	MFRP
Microphthalmia with retinitis pigmentosa	MFRP, PRSS56
Microphthalmia, bilateral	MIP, PAX6
Coloboma, osteopetrosis, microphthalmia, macrocephaly, albinism and deafness	mitftv1 (isoform of MITF), MITF
Microphthalmia, bilateral with sclerocornea	NDP
Pediatric cataract with microphthalmia, microcornea and nystagmus	NHS
Pediatric total cataract with microphthalmia, microcornea and nystagmus
Microcephaly, microphthalmia and cataracts	OCLN
Microphthalmia and coloboma	OLFM2, RAX, TMX3, PAX6, SEMA3E, ABCB6, RBP4
Combined pituitary hormone deficiency, bilateral microphthalmia and agenesis of the left internal carotid artery	OTX2
Combined pituitary hormone deficiency, syndromic microphthalmia and retinal dystrophy
Microphthalmia, ectopic pituitary and growth hormone deficiency
Microphthalmia, pituitary abnormalities and intellectual disability
Microphthalmia, type 5
Microphthalmia, syndromic 5	otx2tv5 (isoform of OTX2)
Syndromic microphthalmia
Bilateral microphthalmia, anophthalmia or coloboma	PAX6
Bilateral microphthalmia, congenital cataract and glaucoma
Bilateral microphthalmia, primary aphakia, coloboma and iris hypoplasia
Microphthalmia, cataracts and nystagmus
Microphthalmia, late-onset keratitis and iris coloboma/aniridia
Anterior segment dysgenesis and microphthalmia	PITX3
Congenital cataract and microphthalmia	PITX3, CRYGC
Non-syndromic A/M	PORCN
Microphthalmia with limb anomalies	PORCN, FNBP4, SMOC1
Mental retardation, microphthalmia, choroid coloboma, microcephaly, renal hypoplasia and spastic paraplegia	PQBP1
Microphthalmia and Peters anomaly	PRR12
Unilateral microphthalmia
Unilateral microphthalmia and Peters anomaly
Microphthalmia with corneal opacification	PXDN
Microphthalmia, sclerocornea, Peters anomaly and aphakia
Microphthalmia and anterior segment dysgenesis	PXDN, GJA8, PAX6, CRYGC, CRYAA
Microphthalmia and diaphragmatic hernia	RARB
Microphthalmia and dystonia
Microphthalmia, syndromic 12
Microphthalmia, isolated, with coloboma 10	RBP4
Microphthalmia, anophthalmia and coloboma disease	RERE, CDON, IQGAP1, CASK, MYO10, TENM3, POLR2A, WNT2B, KIF26B, MICU1, RBP4, FIG4, GDF6
Lenz microphthalmia syndrome	SALL1
Microphthalmia, coloboma and optic nerve hypoplasia	SALL4
Microphthalmia and extensive colobomas of the globes	SIX6
Coffin-Siris syndrome, microphthalmia and small-cell carcinoma of the ovary, hypercalcemic type	SMARCA4
Bosma arrhinia microphthalmia syndrome	SMCHD1
Alzahrani-Kuwahara syndrome and microphthalmia	SMG8
A/M, developmental delay, short stature and ataxic gait	SOX2
A/M, mental retardation, short stature and ataxic gait
A/M, mental retardation, short stature and unsteady gait
Bilateral microphthalmia, microcornea, learning disability and developmental delay
Microphthalmia syndromic 3
Microphthalmia, developmental delay, hearing loss and dysmorphic features
Microphthalmia, iris coloboma, anal atresia and nasal skin tag
Bilateral microphthalmia	SOX2, PAX6
Microphthalmia, anophthalmia and coloboma
Microphthalmia 3	SOX2, RAX
Mild intellectual disability with microphthalmia, coloboma, hypopituitarism, facial dysmorphology and dental anomalies	SOX3
Microphthalmia, syndromic 9	STRA6
Bilateral microphthalmia, congenital cataract, microcephaly, and global developmental delay	TENM3
Microphthalmia, isolated, with coloboma 9
Syndromic microphthalmia 15
Microphthalmia, congenital cataracts and microcephaly	TUBA1A
Microphthalmia, bilateral and coloboma	VSX2
Microphthalmia, cataract and iris abnormality
Anophthalmia and microphthalmia	VSX2, ALDH1A3, PXDN, FOXE3
Microphthalmia and cataract	VSX2, EPHA2
A/M	VSX2, SOX2, RAX, FOXE3, ALDH1A3
Pulmonary hypoplasia, diaphragmatic anomalies, A/M and cardiac defects syndrome	WNT7B
Microcephaly, Microphthalmia, Hyperpigmentation of the skin, Global developmental delay	XPA
Microphthalmia and bilateral chorioretinal coloboma	YAP1
Microphthalmia and bilateral uveal coloboma
Microphthalmia/coloboma	YAP1, KMT2D

A/M: Anophthalmia and microphthalmia; ABCA1: ATP binding cassette subfamily A member 1; ABCA13: ATP binding cassette subfamily A member 13; ABCB6: ATP binding cassette subfamily B member 6; ABCC9: ATP binding cassette subfamily C member 9; AGRN: Agrin; ALDH1A3: Aldehyde dehydrogenase 1 family member A3; ALX1: Aristaless-like homeobox 1; ANOS1: Anosmin 1; AOX1: Aldehyde oxidase 1; ARHGAP6: Rho GTPase activating protein 6; BBS2: Bardet-biedl syndrome 2; BCOR: BCL6 Corepressor; BMP4: Bone morphogenetic protein 4; BRPF1: Bromodomain and PHD finger containing protein; CACNA1F: Calcium channel voltage-dependent, alpha-1F subunit; CACNA2D3: Calcium channel voltage-dependent alpha 2/delta subunit 3; CAPN11: Calpain 11; CAPN15: Calpain 15; CBR4: Carbonyl reductase 4; CD22: CD22 Antigen; CHD7: Chromodomain helicase DNA binding protein 7; CNKSR1: Connector enhancer of kinase suppressor of Ras 1; COL4A1: Collagen type IV alpha 1; COX7B: Cytochrome c oxidase subunit 7B; CRYAA: Crystallin alpha A; CRYBA4: Crystallin beta A4; CRYBB1: Crystallin beta B1; CRYBB3: Crystallin beta B3; CRYGC: Crystallin gamma C; CTNNB1: Catenin beta 1; C12orf57: Chromosome 12 open reading frame 57; DIS3L2: DIS3 like 3'-5' exoribonuclease 2; EFTUD2: Elongation factor Tu GTP binding domain containing 2; EPB41: Erythrocyte membrane protein band 4.1; EPHA2: EPH receptor A2; FAT1: FAT atypical cadherin 1; FERMT1: FERM doamin-containing kindlin 1; FGF20: Fibroblast growth factor 20; FMNL2: Formin like 2; FOXC1: Forkhead box C1; FOXE3: Forkhead box E3; FRAS1: Fraser extracellular matrix complex subunit 1; GAS2L2: Growth arrest-specific 2 like 2; GDF6: Growth/differentiation factor 6; GJA3: Gap junction protein alpha 3; GJA8: Gap junction protein alpha 8; GMPPA: GDP-mannose pyrophosphorylase A; GNAL: G protein subunit alpha L; GRM6: Glutamate receptor metabotropic 6; GRM8: Glutamate receptor metabotropic 8; HCCS: Holocytochrome C Synthase; HECTD2: HECT domain E3 ubiquitin protein ligase 2; KIFC2: Kinesin family member C2; IARS2: Isoleucyl-tRNA synthetase 2; IGFALS: Insulin-like growth factor binding protein, acid-labile subunit; INTS11: Integrator complex subunit 11; IPO13: Importin 13; KIF17: Kinesin family member 17; KMT2A: Lysine-specific methyltransferase 2A; KMT2D: Lysine-specific methyltransferase 2D; KRT31: Keratin 31; LAMB2: Laminin beta 2; LIPH: Lipase H; LRP11: LDL receptor related protein 11; MAB21L1: Mab-21 like 1; MAB21L2: Mab-21 like 2; MAN1A1: Mannosidase alpha class 1A member 1; MCCC1: Methylcrotonoyl-CoA carboxylase 1; MEGF10: Multiple EGF like domains 10; MFSD13A: Major facilitator superfamily domain containing protein 13A; MITF: Microphthalmia associated transcription factor; MSRB2: Methionine sulfoxide reductase B2; MYCBP2: Myc binding protein 2. MYH11: Myosin heavy chain 11; MYO9B: Myosin IXB; NAA10: N-Alpha acetyltransferase 10, NatA catalytic subunit; NDP: Norrin cystine knot growth factor NDP; NDUFB11: NADH-ubiquinone oxidoreductase 1 beta subcomplex 11; NHLRC1: NHL repeat containing protein 1; NHS: NHS actin remodeling regulator; OSGEPL1: O-sialoglycoprotein endopeptidase-like 1; OTX2: Orthodenticle homeobox 2; PALLD: Palladin; PAX6: Paired box 6; PDE6B: Phosphodiesterase 6B; PKHD1L1: PKHD1-like 1; PORCN: Porcupine O-acyltransferase; PRKAG3: Protein kinase AMP-activated non-catalytic gamma 3; PRKG2: Protein kinase cGMP-dependent type II; PUF60: Poly(U) binding splicing factor 60-KD; PXDN: Peroxidasin; P2RY14: Purinergic receptor P2Y, G protein-coupled 14; RAB3GAP2: RAB3 GTPase activating protein non-catalytic subunit; RARB: Retinoic acid receptor beta; RBP4: Retinol binding protein 4; RPGRIP1L: RPGR interacting protein 1 like; SCLT1: Sodium channel and clathrin linker 1; SCN11A: Sodium voltage-gated channel alpha subunit 11; SHH: Sonic hedgehog; SIX6: SIX homeobox 6; SLC18A2: Solute carrier family 18 (vesicular monoamine) member 2; SOX2: SRY-box 2; STRA6: Stimulated by retinoic acid 6; SUMF1: Sulfatase modifying factor 1; TBC1D32: TBC1 domain family member 32; TBCE: Tubulin-specific chaperone E; TENM3: Teneurin transmembrane protein 3; TGFB2: Transforming growth factor beta 2; TUBGCP3: Tubulin gamma complex associated protein 3; TYR: Tyrosinase; VSX2: Visual system homeobox 2; WNT7B: Wnt family member 7B; XKR4: XK-related protein 4; YAP1: Yes1 associated transcriptional regulator; ZP4: Zona pellucida glycoprotein 4.

ENVIRONMENTAL FACTORS

Apart from genetic factors, environmental variables also play a significant role in the development of A/M by influencing embryonic eye formation through different processes. This section discusses the impact of maternal infections, teratogenic exposures, and nutritional deficiencies on A/M.

Maternal infections

The association between maternal infections such as rubella, toxoplasmosis, cytomegalovirus (CMV), and varicella with ocular abnormalities is well-documented[70,71]. Other viruses, such as parvovirus B19[72,73], herpes simplex type 2[74], Epstein-Barr virus[75], and coxsackie A9[76], have been implicated, though with less conclusive evidence. Additionally, infections like influenza, hyperthermia, are associated with both central nervous system malformations and microphthalmia[77-80]. Animal studies provide further support for hyperthermia as a specific cause of A/M, suggesting it may also act as a co-teratogen when combined with other exposures, such as chemicals[77,81].

Teratogenic exposures

Additional risk factors identified in the literature for A/M or other developmental eye anomalies include solvent misuse such as per- and trichloroethylene, toluene and xylene, as well as exposure to X-rays and certain drugs, such as thalidomide, isotretinoin, warfarin, and alcohol[82-85]. Warburg[71] documented that conditions like Goldenhar, CHARGE, and VATER can occur due to maternal exposure to drugs, infections, fever, or radiation, which may disrupt neural crest cell development. Retinoic acid, a regulator of homeobox genes, can cause microphthalmos or colobomas if dysregulated. A study performed by Nichols Barber[86] reported that mouse fetuses heterozygous for anophthalmia had a higher occurrence of eye defects than homozygous normal when their mothers were treated with the teratogen trypan blue. All cases of abnormal eye development involved unilateral anophthalmia, while normal bilateral development was expected in the absence of the teratogen.

Nutritional deficiencies

Maternal nutrition plays a vital role in fetal development, and deficiencies in essential nutrients can significantly impact the development of the eyes. In murine embryos lacking the folate-binding protein 1 gene, about 10%-12% developed anophthalmia or microphthalmia in a folate-deficient environment[87]. A report by Kochhar[88] documented vitamin A, vitamin E and zinc as the essential nutrients for eye development. Nielsen and Carlton[89] reported that vitamin E deficiency is associated with microphthalmia in rabbits, whereas zinc deficient diets in female rats results in visual abnormalities in their offspring[90].

Vitamin A

Vitamin A is essential for normal ocular development, but excessive intake, particularly from vitamin A supplements, can be teratogenic. High vitamin A levels can lead to abnormalities in embryonic development, including microphthalmia[91]. In contrast, inadequate vitamin A during pregnancy may be associated with teratogenic consequences in humans. In a study by Hale[92], a Duroc-Jersey gilt (a pig) when fed with a vitamin A-deficient diet gave birth to eleven anophthalmic piglets. This instance emphasizes the crucial function of vitamin A in normal eye development, since its lack can result in severe congenital abnormalities such anophthalmia[92]. Sarma[93] reported a case of a baby with microcephaly and anophthalmia who survived for only 24 hours, born to a mother suffering from VAD and blindness due to xerophthalmia. Further, another case of a child born with ocular abnormalities, including microphthalmia and coloboma[94] was born to a mother suffering from VAD. World Health Organization recommends a safe maximum dosage during pregnancy is up to 10000 IU per day or 25000 IU per week, beginning after the first 60 days of gestation[95,96].

MECHANISMS INVOLVING EPIGENETIC MODIFICATIONS

Epigenetics refers to heritable changes in gene expression that do not involve alterations to the DNA sequence. Environmental factors such as diet, exposure to toxins, and maternal infections during pregnancy can lead to epigenetic modifications, influencing the risk of congenital anomalies like A/M. These modifications occur through mechanisms such as DNA methylation, histone modification, microRNA and long non-coding RNA regulation, which alter gene activity during fetal development.

DNA Methylation

DNA methylation, primarily occurs at the CpG dinucleotides and involves the addition of a methyl group to cytosine bases, hence leading to gene silencing. This process disrupts the binding of transcription factors and prevents the recruitment of the transcriptional machinery. Methylated cytosines bind to methyl-CpG-binding domain proteins, which recruit repressive complexes that enhance chromatin compaction and suppress transcription. Additionally, methylation can affect mRNA splicing and stability, resulting in fewer mature mRNAs accessible for translation. These pathways result in stable gene silence, which is critical for controlling gene expression during development and maintaining cellular identity. Environmental exposures can cause disruptions in normal methylation processes. For example, a meta-analysis study by Joubert et al[97] reported that maternal smoking during pregnancy has been linked to altered methylation of genes important in eye development, which raises the likelihood of ocular abnormalities in kids. Similarly, folate deficiency, which plays a key role in DNA methylation, can impair ocular development. Studies have shown that reduced folate intake during pregnancy is associated with an increased risk of A/M, likely due to altered DNA methylation patterns that disrupt normal eye morphogenesis[87,98].

Prenatal alcohol exposure can lead to fetal alcohol spectrum disorder (FASD), which manifests as lifelong complications including congenital anomalies, cognitive impairments, and behavioral deficits. Among the abnormalities, microphthalmia is linked to the teratogenic effects of alcohol. While DNA methylation is critical for controlling gene expression, it is mostly unaltered in imprinted areas in FASD. However, maternal supplementation with methyl donors such as folic acid, choline, betaine, and vitamin B12 showed promising results in preventing alcohol-induced developmental problems. Providing proper preconception and prenatal nutritional support is critical for minimizing the incidence of alcohol-related abnormalities, such as microphthalmia, while maintaining normal DNA methylation patterns[99].

Histone modifications

Histone modifications are critical epigenetic mechanisms that regulate gene expression by altering chromatin structure and accessibility of transcriptional machinery to the DNA. These modifications, which include acetylation, methylation, phosphorylation, and ubiquitination, play a significant role in the regulation of genes essential for ocular development. Dysregulation of these processes has been implicated in a variety of congenital eye disorders, including A/M. Sox2 is a transcription factor that plays a pivotal role in early eye development, and its dysregulation due to epigenetic changes such as decreased histone acetylation, can further lead to severe ocular malformations, including microphthalmia. Mutations in genes encoding histone methyltransferases, such as enhancer of zeste homolog 2 (EZH2) and KMT2D, have been linked to developmental disorders involving the eye, including A/M[100]. EZH2 is part of the polycomb repressive complex 2 (PRC2), which catalyzes the methylation of histone H3 at lysine 27 (H3K27), leading to gene silencing. Dysregulation of this process can disrupt the normal pattern of ocular development, as the repression of critical genes involved in eye formation is lifted prematurely or excessively[101]. Aref-Eshghi et al[102] documented that mutations in KMT2D (responsible for methylating H3K4), have been associated with Kabuki syndrome, a disorder that includes ocular anomalies such as coloboma and microphthalmia. Hence, these findings highlight the critical role of histone methylation in regulating gene expression during fetal eye development.

MicroRNAs and Long non-coding RNAs

MicroRNAs (miRNAs) regulate gene expression by binding to mRNAs, inhibiting translation or promoting their degradation. Several miRNAs are involved in eye development, and disrupted regulation of microRNAs can cause ocular malformations. The miR-204 plays a key role in regulating genes important for lens and retinal development. A study by Conte et al[103] showed that miR-204 controls the expression of transcription factors like meis homeobox 2 (Meis2), crucial for eye morphogenesis. Dysregulation of miR-204 has been linked to microphthalmia in the medaka fish (Oryzias latipes) model[103].

Long non-coding RNAs (lncRNAs) regulate gene expression and play critical role in the eye development. Their dysregulation can lead to congenital eye disorders such as A/M. Six3OS lncRNA regulates the SIX3 gene, which is essential for forebrain and early eye development[104]. Rhabdomyosarcoma 2-associated transcript (RMST) is involved in retinal development and interacts with transcription factor Sox2, hence disruption of RMST may affect Sox2 regulation[105].

Noncoding RNAs, including both miRNAs and lncRNAs, regulate crucial pathways during eye development, such as the Wnt and Hedgehog signaling pathways. Dysregulation of Wnt pathway components, influenced by miRNAs like miR-204, can lead to ocular defects such as A/M[103]. Changes in the expression of non-coding RNAs associated with Hedgehog signaling have been noted in microphthalmia models[106].

MANAGEMENT OF A/M

Clinical and surgical management

Individuals with microphthalmia may preserve some visual function, but they are more likely to develop problems such as cataracts, glaucoma, or curable refractive abnormalities. Even with unilateral microphthalmia, it is critical to protect the healthy eye using glasses. Electrodiagnostic studies, such as electroretinography, electro-oculography, and visual evoked potentials, can aid in the diagnosis of optic nerve disorders, retinal dystrophy, or cortical vision abnormalities. Visual acuity should be constantly evaluated, especially in those at risk of retinal dystrophy. Surgery is frequently recommended for serious impairments, like cataracts[107].

Anophthalmia and severe microphthalmia can lead to a reduced orbital size, causing asymmetry. Socket expanders, either hydrophilic or solid, are used from birth to stimulate orbital growth, widen the palpebral fissure, and stretch the conjunctival cul-de-sacs. If an eye is present, a clear prosthesis can help in developing the bony socket and preventing asymmetry. Over time, solid prostheses of increasing size are used to expand the orbital volume, with frequent updates needed in early years to match the contralateral side. Complications may include instability or difficulty with the prosthesis, which are addressed with bespoke, hand-painted designs, and occasionally, orbital implants or socket lining may be required. Once orbital growth is complete in adulthood, changes to prostheses become less frequent[107].

Genetic counseling

Genetic diversity and variable expression complicate the diagnosis and counseling for A/M. While the recurrence risk for siblings was once estimated at 10%-15%, it is now considered lower, though it can vary significantly. Prenatal screening with ultrasound, followed by postnatal assessment, is often used when invasive methods are not possible. Identifying a genetic anomaly can improve counseling and offer options for prenatal or pre-implantation diagnosis, but counseling remains complex due to phenotypic variability and potential mosaicism.

CHALLENGES IN STUDYING GENE-ENVIRONMENT INTERACTIONS

Studying gene-environment interactions in A/M involves several challenges. The major challenge is to separate particular environmental exposure and correlating it to genetic predispositions, as variables i.e., maternal infections, dietary inadequacies, and toxin exposure frequently coexist, confounding the evaluation of their individual effects[108]. Because these situations are rare, obtaining significant sample size for rigorous statistical analysis is challenging. Furthermore, examining epigenetic regulation, which may explain these associations, requires high throughput techniques such as DNA methylation profiling and histone modification analyses. These approaches are costly and require large cohorts to get statistically significant results[97]. Furthermore, sometimes animal models may not fully reflect human ocular development, limiting the findings' relevance due to genetic variations and environmental exposure[97,109].

EMERGING RESEARCH IN A/M

Advances in genetic screening

Whole-exome sequencing (WES) and whole-genome sequencing (WGS) have revolutionized research in A/M, resulting in the discovery of new genes and pathways involved in eye development. These high throughput technologies helped researchers to identify rare genetic mutations that previously remained undetected due to traditional approaches, therefore, offering more insight into the complex genetic architecture of A/M[43,110].

WES has been useful in identifying disease causing mutations in genes such as SOX2, OTX2, and VSX2, responsible for early fetal eye development[43,110]. Furthermore, WGS has higher benefits than WES, as it can detect structural variants, variants present in the non-coding regions, and regulatory factors that might contribute to congenital eye anomalies. WGS can also identify copy number variants (CNVs) that alter genes critical for eye development, assisting in the elucidation of new pathogenic pathways. As our understanding of genetic diversity grows, researchers are better able to examine how these interactions may impact the development of A/M. Future research may improve the significance of WES and WGS, especially as sequencing prices fall and larger-scale studies become feasible[108].

Contributions of animal models

Animal models, especially mice and zebrafish, have greatly advanced our knowledge of A/M. These animal models share similar eye morphology with humans and can be genetically modified, enabling researchers to investigate the role of specific genes in eye development. Because of the similarities in ocular development and physiology between mice and humans, many mouse lines exhibiting microphthalmia have been created, primarily by disrupting genes associated with the condition in humans. These genes include Sox2, Otx2, Rax, Vsx2, Pax6, Stra6, Foxe3, Bmp4, Bmp7, sparc-related modular calcium-binding protein 1 (Smoc1), Shh, porcupine o-acyltransferase (Porcn), forkead box C1 (Foxc1), fraser extracellular matrix complex subunit 1 (Fras1), fras1-related extracellular matrix protein 1 (Frem1), tectonic family member 2 (Tctn2), collagen type IV alpha 1 (Col4a1), TBC1 domain family, member 32 (Tbc1d32), protease, serine 56 (Prss56), peroxidasin (Pxdn), paired-like homeodomain transcription factor 2 (Pitx) 2, Pitx3, Mitf, alpha A crystallin (Cryaa), Frem2, retinitis pigmentosa GTPase regulator-interacting protein (Rpgrip1), SMG9 nonsense-mediated mRNA decay factor (Smg9), sorting nexin 3 (Snx3), dystroglycan 1(Dag1),H6 family homeobox 1 (Hmx1), arginine-glutamic acid dipeptide repeats (Rere), and RAS-associated protein (Rab18) (https://informatics.jax.org). Similarly, other animal models i.e., Zebrafish (Otx2, Rax, Pax6, Stra6, Foxe3, Gdf6, Shh, Mab21 Like 2 (Mab21l2), patched 2 (Ptch2), membrane-type frizzled-related protein (Mfrp), Pitx2, Six3, Rere) (https://zfin.org) and Xenopus (Pax6) (https://xenbase.org) have been created to study the role of different genes in eye development (microphthalmia and anophthalmia).

Stem cell and regenerative medicine research

Stem cell research offers potential for treating A/M. Induced pluripotent stem cells (iPSCs) can generate retinal cells and other eye tissues, providing opportunities for regenerative medicine[111]. The iPSC-based models help study disease-specific mutations and their impact on eye development. A human induced pluripotent stem cell (hiPSC) line UCLi013-Awas established from fibroblasts obtained from a 34-year-old donor with severe microphthalmia and aniridia, carrying a heterozygous missense mutation in the PAX6 gene [c.372C>A, p.(Asn124 Lys)], confirmed via Sanger sequencing. This cell line may offer a valuable platform for studying microphthalmia and aniridia, identifying therapeutic targets, and screening potential drugs[112]. Similarly, Eintracht et al[113] used iPSC-derived OVs from two microphthalmia patients and healthy controls. RNA sequencing identified upregulated apoptosis and ECM genes. Increased expression of LUM, nidogen, and collagen type IV suggested ECM overproduction. The authors further reported that pharmacological inhibition of caspase-8 with Z-IETD-FMK decreased apoptosis in one patient model, indicating a possible therapeutic strategy. Though still in its early stages, stem cell therapy may one day restore vision or regenerate ocular structures in individuals with severe eye malformations[114].

Translating genetic discoveries into clinical applications

The rapid advancements in genetic and genomic research have significantly enhanced the diagnostic landscape for A/M. Clinical applications of genetic discoveries in A/M include molecular diagnostics, personalized therapeutic interventions, and emerging gene therapies.

Genetic testing for precision diagnosis: WES and WGS have revolutionized the ability to diagnose A/M, particularly in non-syndromic cases where clinical features alone are insufficient. CNV analysis and chromosomal microarray are instrumental in detecting larger structural variations associated with A/M. In a mixed microphthalmia, anophthalmia and coloboma (MAC) cohort, Harding et al[115] using targeted gene panels, WGS, and microarray comparative genomic hybridization, identified pathogenic mutations in approximately 33% of cases, with a diagnostic yield that was consistent for both unilateral and bilateral cases. Systemic associations were observed in 34% of patients, emphasizing the need for a multidisciplinary approach. Imaging techniques such as magnetic resonance imaging and B-scan ultrasound are crucial for evaluating ocular structure and associated abnormalities. Novel findings, including the association of EPHA2 with microphthalmia and FOXE3 with hearing loss and kidney anomalies, contribute to expanding current knowledge. Harding et al[115] underscored the importance of comprehensive phenotyping, genetic testing, and individualized management to improve patient outcomes.

The integration of these genetic tools into clinical practice allows for earlier and more accurate diagnosis, guiding genetic counseling and reproductive decision-making for affected families. WES is increasingly used in prenatal diagnosis when fetal anomalies are detected on ultrasound but conventional genetic tests like karyotyping and microarray fail to provide a diagnosis. Studies have shown that WES improves the diagnostic yield in such cases, with rates ranging from 6.2% to 57%[116-118]. Prenatal WES has been successfully used in detecting SOX2, OTX2, and RAX mutations, leading to early intervention and improved perinatal care[119]. A study investigating five cases of fetal ultrasound abnormalities using WES at a single institution reported a diagnostic yield of 80%, with four out of these five cases yielding a confirmed genetic diagnosis. In one of these cases, anophthalmia and a choroid plexus cyst were identified at 19 weeks of gestation[120]. Given the known genetic associations, WES confirmed a de novo SOX2 pathogenic variant, a known cause of syndromic anophthalmia[41]. The pregnancy was terminated before WES results, and autopsy confirmed anophthalmia. This represents a rare instance of prenatal diagnosis of syndromic microphthalmia[120].

Personalized therapeutic approaches: With advances in molecular biology, personalized medicine for A/M is becoming a possibility. The identification of patient-specific mutations enables targeted interventions, including pharmacological treatments that modulate affected developmental pathways. For instance, retinoic acid derivatives have been explored for PAX6 and ALDH1A3-related microphthalmia, given their role in ocular morphogenesis[14,121].

Gene therapy and stem cell-based interventions: Gene therapy for A/M is currently an area of active research. IPSCs derived from patients with microphthalmia have been used to generate retinal organoids, offering a potential source for regenerative therapies[122]. The hiPSCs with a homozygous VSX2 mutation (p.Arg200Gln) associated with microphthalmia were differentiated into early optic cups, showing normal initial development but disrupted optic cup formation due to reduced proliferation and increased RPE differentiation[57]. RNA-seq revealed upregulation of Wnt/transforming growth factor signaling (WNT11, BMP8A) and downregulation of fibroblast growth factor (FGF) signaling (FGF19) in mutant vesicles[57]. Further studies confirmed dysregulated Wnt signaling and abnormal VSX2/MITF co-expression, disrupting neural retina/RPE differentiation in microphthalmia[56]. Wnt inhibition partially rescued neuroretina formation but failed to restore the RPE layer, while Wnt activation in wild-type vesicles replicated the microphthalmia phenotype[56].

Global health disparities in A/M recognition and management

A/M prevalence and clinical outcomes vary significantly across regions, particularly in low-resource settings, where early diagnosis and genetic testing remain largely unavailable. While high-income countries benefit from advanced diagnostic tools such as WGS and prenatal imaging, many low-income and middle-income countries (LMICs) rely solely on clinical examination, leading to underdiagnosis and mismanagement[123,124]. Due to limited access to healthcare, advanced prenatal clinics and unaffordability, early detection of MAC spectrum disorders remains a challenge in LMICs. Severe microphthalmos and anophthalmos with or without cysts which can easily be detected by prenatal ultrasound are often missed due to lack of adequate healthcare[125,126].

The largest study of MAC disorders published was conducted as a collaborative study in three tertiary eye care centers from North India to understand the clinical spectrum of non-syndromic MAC disorders in India in a one-year study period[127]. Total 545 children (0-18 years of age) were included in the study; however, despite the enormous disease burden, genetic investigations were not carried out to decipher the molecular causation. This is due to the steep costs of genetic investigations in LMICs and the lack of support by insurance agencies in carrying out personalized genetic tests. A trend of segregation of severe types of MAC was seen in a particular region where, co-incidentally, VAD related corneal disease was common. Nutritional deficiencies such as VAD are considered a significant public health concern, with a relatively high prevalence, where studies report a considerable percentage of the population suffering from subclinical or clinical VAD, often linked to inadequate dietary intake of vitamin A-rich foods[128]. Considering the evidence in literature regarding the interplay between Vitamin A pathway genes and MAC disorders[94], a relationship between the maternal VAD and incidence of MAC could be hypothesized (Subhedkar et al, data unpublished). Both VAD and MAC are more common in the lower socio-economic classes[129,130] making them a serious health concern in LMICs. The MAC disorders have also been found to be more common in rice-dependent communities and consanguineous populations[131,132].

A study in Colombia examined the causes of eye loss among patients, revealing that over 60% of cases were from low socioeconomic backgrounds. The study highlighted a significant gap in healthcare coverage, as the national health system did not cover expenses related to ocular prostheses. Additionally, more than 75% of patients had been using the same ocular prosthesis for over a decade, reflecting a lack of follow-up care and limited resources for maintenance[133].

These cases underscore the critical need for improved access to specialized healthcare, early diagnostic tools, and comprehensive rehabilitation services in LMICs.

Efforts to bridge the gap in A/M management include: (1) Low-cost genetic testing initiatives: Programs such as the Human Heredity and Health in Africa (H3Africa) project aim to expand access to affordable genetic testing[134]; (2) Telemedicine and artificial intelligence-based diagnostics: Artificial intelligence-driven imaging platforms are being developed to facilitate remote diagnosis and genetic counseling in underprivileged areas[135]; and (3) Community-based genetic screening: In countries such as India and Brazil, public health initiatives are integrating community-based genetic screening programs, reducing the burden of undiagnosed congenital conditions[136].

FUTURE DIRECTIONS IN A/M RESEARCH

Integrating genomic and environmental data

Future studies on A/M should integrate genetic and environmental data to investigate their complex relationships. Building comprehensive databases with genomic, transcriptomic, proteomic, and environmental information would allow researchers to investigate gene-environment interactions more effectively. More epidemiological studies should be performed that focusses on maternal exposures during pregnancy such as infections, drugs, and dietary variables. These studies may give crucial data for developing public health policy to protect at-risk populations from these hazards. This information might lead to customized medical approaches, allowing early identification of high-risk people and individualized therapy tactics that target both genetic and environmental effects[108].

Development of preventative strategies

The future of preventing A/M depends on integrating genetic counseling with public health measures that address environmental risks. Genetic counseling for A/M is difficult due to the large range of associated genes and phenotypic heterogeneity. The key gene found is SOX2, and most mutations arise de novo. It is difficult to predict recurrence risk, especially when there is mosaicism or varied penetrance in monogenic cases. If the mechanism of inheritance is identified, targeted counseling can be offered, with a 10%-15% risk to siblings when no apparent reason or family history is evident[43,137]. Chromosomal abnormalities often present with distinct syndromes, and risk to siblings depends on whether the parents carry any chromosomal rearrangements[1,110]. Furthermore, advances in prenatal genetic testing, especially non-invasive prenatal testing, may allow for early diagnosis of certain mutations, aiding informed decision-making during pregnancy[43].

Public health strategies aimed at reducing environmental risks are also crucial. For example, improving maternal health through nutritional supplementation-especially with folic acid, which has been shown to reduce the risk of neural tube defects, may also reduce ocular malformations-and thus could help decrease the incidence of A/M[87]. In addition, public health campaigns aimed at reducing exposure to teratogens, such as alcohol, isotretinoin, and infections like CMV and rubella, can play an important role in prevention. Several years ago in England, there was public concern about potential clusters of A/M cases. The pesticide benomyl, and later its derivative carbendazim, were suspected of being linked to these clusters[138].

Prospects for therapeutic interventions

Recent advances in gene therapy, stem cell research, and regenerative medicine offer promising avenues for treating A/M. While these approaches are still in the early stages of development, they hold great potential for the future. Gene therapy, for instance, has shown success in treating retinal degenerative diseases, and similar techniques may eventually be adapted to target genetic defects responsible for A/M[114]. Correcting mutations in genes like SOX2 or OTX2 through gene editing technologies like clustered regularly interspaced short palindromic repeats-Cas9 could offer a future cure for these conditions[108]. Stem cell research also holds promise, particularly through the use of iPSCs to generate retinal cells and other ocular tissues for transplantation. Stem cell-based therapies could potentially replace lost or malformed tissues in patients with A/M, offering a regenerative approach to treatment[111]. However, significant challenges remain, including ensuring the safety and efficacy of these treatments, as well as addressing ethical concerns surrounding genetic manipulation and stem cells use.

While these therapeutic interventions offer exciting possibilities, more research is needed to fully understand their potential and to develop safe, effective treatments for A/M. Ethical considerations, such as access to these advanced therapies and the long-term consequences of gene editing, will also need to be addressed as the field progresses[114].

CONCLUSION

A/M, as severe congenital eye disorders have garnered significant attention due to their complex etiologies involving both genetic and environmental factors. Over the last decade, advances in genetic screening techniques, such as whole-exome and WGS, have significantly expanded our understanding of the genetic basis of A/M. Mutations in key developmental genes, including SOX2, OTX2, RAX, and VSX2, have been identified as primary contributors to the pathogenesis of these conditions[43,110]. Additionally, emerging evidence highlights the role of non-coding RNAs, such as miR-204 and Six3OS, in regulating critical pathways during eye development, further emphasizing the complexity of genetic regulation in A/M[103,104]. At the same time, maternal environmental exposures-including infections, teratogens, and nutritional deficiencies-have been implicated in the development of A/M. Studies have shown that maternal infections like rubella, CMV, and toxoplasmosis, as well as exposure to alcohol, isotretinoin, and folate deficiencies, can increase the risk of ocular malformations[38,87]. These findings underscore the critical need for a comprehensive approach that integrates research at both genetic and environmental level. By focusing on both prevention through public health measures and innovative therapeutic interventions, we can hope to improve outcomes for individuals affected by these rare but severe ocular malformations.