Molecular Vision 2026; 32:1-20 <http://www.molvis.org/molvis/v32/1>
Received 19 August 2025 | Accepted 18 January 2026 | Published 20 January 2026

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Exploring the molecular basis of microphthalmia and anophthalmia: Insights from an Egyptian cohort

Gehad Elmakkawy,1 Amira Nabil,1 Karim Nabil,2 Asmaa Kenawy Amin,1 David Maskill,3 Manir Ali,3 Daniel Schorderet,4 Nader Bayoumi,2 Nihal Shakankiri,2 Ebtesam Abdalla1

Last two authors contributed equally to this work.

1Department of Human Genetics, Medical Research Institute, Alexandria University; 2Department of Ophthalmology, Faculty of Medicine, Alexandria University, Egypt; 3Leeds Institute of Medical Research, University of Leeds, St. James's University Hospital, Leeds, UK; 4School of Life Science, Ecole Polytechnique Fédérale de Lausanne (EPFL), and University of Lausanne, Lausanne, Switzerland

Correspondence to: Ebtesam M. Abdalla, Human Genetics Department, Medical Research Institute, Alexandria University, Egypt. email: Ebtesam.nasr@alexu.edu.eg

Abstract

Purpose: The aim of this study was to gain insight into the molecular spectrum of anophthalmia and microphthalmia (A/M) in the Egyptian population.

Methods: We studied a cohort of 34 patients from 31 unrelated families affected by the A/M spectrum. All patients underwent a thorough clinical examination, ophthalmological assessment, and genetic testing including conventional karyotyping and exome sequencing (ES).

Results: Chromosomal anomalies were identified in six patients. ES was performed on the remaining cases, revealing potentially causative variants in 13 families. The implicated genes were SOX2, OTX2, CHD7, HMX1, PRR12, ATOH7, ZBTB11, B3GALNT2, GCNT2, DPH1, GJA8, FRAS1 and UBE3B. Among the variants, six were classified as pathogenic, five as likely pathogenic, and two as variants of uncertain significance. Notably, a DPH1 pathogenic variant was identified in a patient with bilateral severe microphthalmia, representing a novel phenotype. Additionally, we report the fifth family diagnosed with oculo-auricular syndrome.

Conclusions: Our findings confirm that genetic factors are a predominant cause of both syndromic and non-syndromic A/M and underscore the value of ES in uncovering the molecular basis of this spectrum. By reporting novel variants and unusual phenotypes within our cohort, we contribute to expanding both the mutational landscape and the phenotypic spectrum of A/M associated syndromes.

F

Introduction

Anophthalmia and microphthalmia (A/M) are severe congenital eye defects that fall within a spectrum of ocular anomalies, which may also include coloboma. Several developmental and regulatory genes play critical roles in eye formation during embryogenesis, and disruptions in these genes can result in either the complete absence of the eye (anophthalmia) or the formation of a small, underdeveloped eye (microphthalmia) [1-3]. A/M can occur as an isolated anomaly (non-syndromic) or as part of a broader phenotype involving extraocular anomalies (syndromic). Syndromic A/M represents the majority of cases and tend to have a higher rate of identifiable genetic causes and diagnostic yield [4-6].

The genetic basis of A/M has been extensively studied, revealing a complex interplay of single-gene mutations and chromosomal abnormalities. Nearly 100 genes have been implicated in the pathogenesis of A/M [7]. Additionally, conventional cytogenetic analysis can detect significant chromosomal aberrations in up to 15% of affected individuals [5,8,9]. The considerable genetic heterogeneity of A/M, which is further complicated by variable expressivity, incomplete penetrance, and overlapping phenotypes, poses significant challenges for diagnosis and genetic counseling.

Understanding the specific genetic landscape of A/M in any given population is essential for improving diagnostic accuracy and guiding patient care. The paucity of genetic studies on A/M makes it impossible to accomplish these objectives, underscoring the necessity for more research. Thus, the present study aimed to investigate the molecular characteristics of A/M and evaluate the role of ES in diagnosing a cohort of Egyptian patients affected by this spectrum of ocular anomalies.

Methods

Patient recruitment

Ethical approval was obtained from the Ethics Committees of both Medical Research Institute and the Faculty of Medicine, Alexandria University. The legal guardians of all participants provided written informed consent, aligned with the principles outlined in the Declaration of Helsinki. In this cross-sectional cohort study, 34 patients with A/M spectrum disorders were recruited between June 2021 and December 2023.

The inclusion criteria encompassed individuals of any age and gender who had been clinically diagnosed with A/M. Patients with a documented history of teratogenic exposure were excluded from the study.

After obtaining detailed medical and genetic history, a comprehensive clinical evaluation was performed. Whole blood samples were collected from the probands and their parents, and whenever possible from their healthy (unaffected) siblings.

Cytogenetic analysis

Karyotyping was performed for all the enrolled cases using the G-banding technique, following the method described previously [10]. Cases with an abnormal karyotype were excluded from further analysis.

Exome sequencing (ES)

Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Exome capture was performed using the xGen Exome Research Panel v2 (Integrated DNA Technologies, Coralville, IA) and sequencing was performed on the NovaSeq 6000 (Illumina, San Diego, CA). Sequencing reads were aligned to the Genome Reference Consortium Human Build 37 (GRCh37) and the Revised Cambridge Reference Sequence (rCRS) for the mitochondrial genome. This process generated a mean depth of coverage of 146.08x across the 34,366,188 bases of the captured region, covering approximately 99.3% of the RefSeq protein- coding regions. Additionally, 98.90% of the targeted bases were covered to a depth of ≥20×.

Sequencing data analysis and variant interpretation was performed using EVIDENCE v4.3 (Seo et al., 2020). Variant Effect Predictor (VEP, v104.2) was used for variant annotation. Variants were prioritized based on the guideline recommended by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology [11].

A variant was considered potentially damaging if it was predicted to alter the protein structure or function by in silico tools such as SIFT, Mutation Taster2, PolyPhen-2, and Align GV-GD, or if it resulted in stop-gain variants, frameshift insertions/deletions, or alterations at canonical splice sites. Additionally, variant rarity was assessed using public databases including 1000 Genome Project and gnomAD (v4.1.0), with a threshold allele frequency of ≤1%. Sanger sequencing test was used to validate candidate variants and confirm segregation on DNA from family members.

Results

The study cohort comprised 34 patients with A/M, from 31 unrelated Egyptian families. Their ages ranged from 1 day to 18 years. Parental consanguinity was found in 13 families (41.9%) while a positive history of familial recurrence was documented in 7 families (22.6%).

Clinical findings

Regarding the ocular phenotype, 52.9% (18/34) of the patients had bilateral microphthalmia, 23.5% (8/34) had unilateral microphthalmia, 14.7% (5/34) presented with bilateral anophthalmia, and 8.8% (3/34) had mixed forms of A/M. In 21 patients (61.7%), the A/M phenotype was complex and was accompanied by additional ocular findings (Table 1). Within the cohort, 19 patients (55.9%) exhibited extraocular manifestations and were classified as having syndromic A/M (P22-P34 in Table 1, in addition to P1-P6 in Appendix 1), whereas the remaining 15 cases (44.1%) had isolated A/M (P7-P21 in Table 1).

Cytogenetic findings

Conventional karyotyping identified chromosomal abnormalities in six patients; four cases of trisomy 13 (Appendix 2), one with trisomy 18, and one with a reciprocal translocation t(3;11; q27;p11.2). The demographic, clinical and cytogenetic data of these patients are summarized in Appendix 1.

Molecular findings

After excluding the six patients with chromosomal abnormalities, ES was conducted on 28 patients from 25 unrelated families. In 13 families (13/25; 52%), potential candidate variants were identified, each in a distinct gene: SOX2 (SRY-box transcription factor 2, Gene ID: 6657), OTX2 (orthodenticle homeobox 2; GeneID 5015), CHD7 (chromodomain helicase DNA binding protein 7; GeneID 55636), HMX1 (H6 family homeobox 1; GeneID 3166), PRR12 (proline rich 12; GeneID 57479), ATOH7 (atonal BHLH transcription factor 7; GeneID 220202), ZBTB11 (zinc finger and BTB domain containing 11; GeneID 27107), B3GALNT2 (beta-1,3-N-acetylgalactosaminyltransferase 2; GeneID 148789), GCNT2 (glucosaminyl (N-acetyl) transferase 2 (I blood group); GeneID 2651), DPH1 (diphthamide biosynthesis 1; GeneID 1801), GJA8 (Gap Junction Protein Alpha 8) with human GeneID 2703), FRAS1 (Fraser extracellular matrix complex subunit 1; GeneID 80144) and UBE3B (ubiquitin protein ligase E3B; GeneID 89910; Table 1). Out of these variants, seven had not been previously reported and were absent from population databases. Based on ACMG guidelines, 11 variants were classified as pathogenic or likely pathogenic (P/LP), while two were considered variants of uncertain significance (VUS). In the remaining 12 families, no clinically significant variants could be identified (Figure 1). Notably, positive molecular findings were detected in eight out of 13 families with syndromic A/M (61.5%) and in 5 out of 12 with non-syndromic A/M (41.6%), highlighting a higher diagnostic yield in syndromic cases.

The pedigrees of the families with positive molecular findings with their associated genetic data are demonstrated in Figure 2.

Positive molecular findings

A novel heterozygous variant in OTX2 (Microphthalmia, syndromic 5; OMIM610125)-- A 5-year-old male patient (F7/P7), born to healthy, non-consanguineous parents, presented with left eye anophthalmia and right eye microphthalmia with Peters anomaly. Brain and orbit MRI showed left anophthalmia and optic nerve hypoplasia, while the overall brain structure and pituitary gland appeared normal. ES identified a heterozygous likely pathogenic missense variant in OTX2: c.278G>C, p.(Trp93Ser). This variant had not been previously reported in the literature and was absent from the gnomAD database.

A novel heterozygous variant in PRR12 (Neuro-ocular syndrome; OMIM619539)-- A heterozygous pathogenic nonsense variant in PRR12:c.3625C>T, p.(Arg1209Ter), was identified in a 3-month-old male infant (F8/P8) exhibiting unilateral microphthalmia with optic disc and chorioretinal coloboma. PRR12 has been associated with an autosomal dominant (AD) neuro-ocular syndrome [12].

A homozygous VUS in GCNT2 (Cataract 13 with adult i phenotype; OMIM116700)-- We identified a homozygous GCNT2 variant: c.1154G>A, p.(Arg385His), in 10- and 8-year-old female siblings (F9/P9&10), presenting with bilateral congenital cataracts and microphthalmia. The younger sister also exhibited nystagmus and strabismus. The identified VUS, was previously described in two siblings with congenital cataracts and nystagmus [13].

A novel homozygous likely pathogenic variant in ATOH7 (PHPV, AR; OMIM221900)-- A homozygous ATOH7 missense variant (c.254C>T), was identified in 5- and 3-year-old similarly affected sisters (F10/P11&12; Figure 3), who were diagnosed with persistent fetal vasculature (PFV). Ophthalmic evaluation revealed bilateral inoperable PFV associated with total retinal detachment, subretinal hemorrhagic effusion, scattered corneal scarring and bilateral congenital cataract, which was not dense enough to obscure fundus examination. Orbital MRI demonstrated bilateral microphthalmia.

A heterozygous pathogenic variant in GJA8 (Cataract 1, multiple types; OMIM116200)-- A heterozygous GJA8 missense variant (c.134G>C) was identified in a 13-year-old girl (F11/P13) with bilateral congenital cataract, glaucoma, bilateral microcornea, and microphthalmia. A similar ocular phenotype was observed in her father, indicating an AD inheritance pattern.

A heterozygous VUS in SOX2 (Syndromic microphthalmia-3 (OMIM206900)-- A heterozygous VUS in SOX2: c.178G>C, p.(Ala60Pro) was identified in a stillborn infant (F19/P22) displaying bilateral anophthalmia, esophageal atresia, tetralogy of Fallot and bilateral talipes equinovarus. The variant is a missense change with a high pathogenicity prediction score and was absent from the gnomAD database. The above-mentioned congenital malformations were revealed during a routine prenatal US examination and verified by fetal MRI.

A heterozygous likely pathogenic variant in CHD7 (CHARGE syndrome; OMIM214800)-- In a 16-year-old female (F20/P23) presenting with left anophthalmia, right eye microcornea, inferior iris coloboma, inferior chorioretinal coloboma and high myopia, ES identified a heterozygous CHD7 splicing variant in: c.5405–17G>A, p.(?). Clinical examination revealed dolichocephaly, broad forehead with receding anterior hairline, facial asymmetry, left anophthalmia, right eye coloboma with nystagmus, a bulbous nose with flattened tip, columella extending below alae nasi, wide mouth with thin upper lip and low-set malformed ears. Furthermore, abdominal and pelvic ultrasound revealed agenesis of the uterus and both ovaries.

A Novel HMX1 variant in the fifth family of the Schorderet-Munier-Franceschetti ‘Oculoauricular syndrome’ (OMIM612109)-- Molecular testing revealed a novel homozygous HMX1 frameshift variant: c.570_571del, p.(Glu191Argfs*34) in a 6-year-old female (F21/P24) presenting with bilateral microcornea and dense bilateral congenital cataract. The patient exhibited average physical growth, while both motor and intellectual development were significantly delayed. Craniofacial dysmorphism included long face, deeply set eyes, convergent strabismus, scanty eyebrows, and protruding, low-set ears with lobule aplasia and a narrow external acoustic meatus (Figure 4). The patient also displayed excessive sweating in cold weather. Ophthalmic examination, including B-scan ocular ultrasonography, revealed microphthalmia with marked microcornea, sclerocornea and adherent central corneal leukoma in the right eye and total corneal leukoma, aniridia, and PFV in the left eye. Appendix 3 summarizes the clinical and molecular findings of this patient compared to the previously reported cases of Schorderet-Munier-Franceschetti oculoauricular syndrome.

Molecular and phenotypic expansion of ZBTB11- related neurodevelopmental disorder (OMIM618383)-- A homozygous splicing variant in ZBTB11:c.1623+2T>G was identified in a 7-month-old infant (F23/P26) born to consanguineous healthy parents. The patient presented with microcephaly, plagiocephaly, scanty hair, receding frontal hairline, thin lips, high arched palate, and low set, cupped ears. Ophthalmic assessment revealed bilateral lamellar cataract, attenuated retinal vessels, and bilateral microphthalmia. Neuroimaging showed bilateral dense cerebral calcifications, hydrocephalus, agenesis of corpus callosum and a hypoplastic cerebellar vermis.

A Novel homozygous pathogenic variant in B3GALNT2 (muscular dystrophy-dystroglycanopathy, type A, 11; # 615,181)-- A homozygous B3GALNT2 nonsense variant: c.1338G>A, p.(Trp446*) was identified in a 7-day-old male (F24/P27) presenting with congenital hydrocephalus and left microphthalmia. The consanguineous parents reported a similarly affected sibling. Brain MRI revealed cobblestone type II lissencephaly, marked supratentorial hydrocephalus, hypoplasia of the cerebellar hemispheres and vermis, and a z-shaped hypoplastic pons. Ophthalmic examination showed optic disc hypoplasia in the right eye, and severe microphthalmia with reduced intraocular pressure in the left eye. Electroencephalography demonstrated left temporal epileptiform discharges and creatine kinase levers were elevated.

A homozygous variant in UBE3B (Kaufman oculocerebrofacial syndrome; OMIM244450)-- A homozygous pathogenic variant in UBE3B: c.1956+1G>A, p.(?), was identified in a 6-month-old female (F25/P28) with left anophthalmia and severe right microphthalmia. She also exhibited microcephaly, frontal bossing and sparse eyebrows. Additionally, congenital contractures, hypoplastic distal phalanges of the index fingers, and hypoplastic nails of fingers and toes, inverted hypoplastic nipples and an accessory nipple were noticed. The patient was born to consanguineous parents, who reported a similarly affected child that died in early infancy. The identified UBE3B variant is a strong splice-alternating variant that causes exon skipping, ultimately disrupting the reading frame and impairing protein function.

A homozygous variant in FRAS1 (Fraser syndrome 1; OMIM219000)-- In a 6-month-old female (F26/P29), presenting with syndromic microphthalmia, a homozygous FRAS1 frameshift variant: c.2376del, (p.Ser793AlafsTer177) was detected. The patient displayed left cryptophthalmos, right upper eyelid coloboma, sclerocornea, hypoplastic nares with nares coloboma and bilateral stenotic auditory meatus. Bilateral incomplete syndactyly between the 3rd through 5th fingers, as well as complete syndactyly of all toes were also noted. The external genitalia showed hypoplastic labia majora, absent vaginal opening and clitoromegaly. Karyotyping revealed normal female karyotype. MRI of the orbit showed left microphthalmia with a cyst, decreased optic nerve diameter, and hypoplasia of the left optic chiasma. Abdominal US demonstrated agenesis of the right kidney.

The possible structural effects of the three novel missense variants identified in the cohort were predicted by SWISS MODEL and Hope [14,15], and demonstrated in Appendix 4.

Discussion

Anophthalmia and microphthalmia (A/M) represent a spectrum of ocular developmental abnormalities. A/M is a highly heterogeneous condition, with nearly 100 associated genes identified to date [3]. Establishing genotype-phenotype correlations is essential for guiding patient care and genetic counseling.

Among the 28 patients (from 25 unrelated families) subjected to ES in the current study, causative variants were identified in 13 families (13/25; 52%). This relatively high diagnostic yield may be attributed, in part, to the high rate of parental consanguinity in our cohort, which increases the likelihood of AR inheritance. Our findings are comparable to those of the limited number of studies reporting the diagnostic yield of ES in ocular development disorders [7,16-19]. The higher diagnostic rate reported by Matias-Perez et al. [20], however, may be due to the predominance of bilateral and severe A/M phenotypes in their cohort.

This study revealed several noteworthy findings, including: the identification of novel variants, the association of A/M with genes not previously linked to ocular anomalies, and the diagnosis of patients with ultra-rare genetic disorders. Notably, seven out of the variants identified in our cohort were novel; all absent from gnomAD. Furthermore, we observed instances of phenotypic expansion with A/M emerging as a newly recognized clinical feature in patients carrying DPH1 and GCNT2 variants (Appendix 5). We also report bilateral microphthalmia and congenital heart disease as novel features in a patient with bi-allelic ZBTB11 variants, thus broadening the phenotype of this ultra-rare neurodevelopmental disorder.

Among this cohort, a novel heterozygous LP variant in OTX2 (c.278G>C, p.Trp93Ser) was identified in a patient with A/M and Peters anomaly (F7/P7). The transcription factor OTX2 is known to play a crucial role in ocular, craniofacial, and pituitary development. Pathogenic variants in OTX2 have been associated with diverse ocular phenotypes [21-24]. Extraocular anomalies - including variable congenital hypopituitarism and ID, growth retardation and deafness - have occasionally been reported [25-27]. In contrast to these reports, the case described here exhibited normal development and growth, with no apparent extraocular anomalies. These inconsistencies may be attributed to the variable expressivity of OTX2 mutations or to the possibility of late-onset pituitary dysfunction.

Another novel genetic defect – a heterozygous nonsense variant in PRR12 (c.3625C>T, p.Arg1209*) - was identified in an infant (F8/P8) presenting with unilateral microphthalmia accompanied by disc and chorioretinal coloboma. PRR12 is predicted to be highly intolerant to loss-of-function (LOF) variants [28], and its haploinsufficiency has been linked to a neuro-ocular syndrome, which encompasses a wide range of clinical features, including consistent neurodevelopmental impairment in all reported cases and variable ocular anomalies [12]. The isolated complex microphthalmia observed in this study is consistent with previous reports [29]. However, intellectual capacity could not be evaluated due to the patient’s young age. As such, ongoing follow-up and reassessment are necessary.

Moreover, a homozygous VUS in GCNT2 was identified in two similarly affected sibs (F9/P9&10) with bilateral microphthalmia and cataract. Biallelic variants in GCNT2 have been associated with cataract 13 with adult i phenotype. To the best of our knowledge, this is the first report of a homozygous GCNT2 variant associated with A/M. One possible explanation for this novel phenotype could be the limited number of reported GCNT2 variants until now. Therefore, additional cases and functional studies are needed to provide further evidence for reclassifying this variant and to confirm the potential association of GCNT2 and A/M.

Although heterozygous GJA8 mutations are known to be associated with isolated cataracts 1, multiple types, we identified a heterozygous GJA8 pathogenic variant (c.134G>C) in a patient with congenital cataract, glaucoma and microphthalmia (F11/P13). Ma et al. (2016) also reported A/M anomalies in patients carrying GJA8 variants [30]. More recently, GJA8 variants have been implicated in a rare condition not currently listed in the OMIM database, termed ‘familial acorea-microphthalmia-cataract syndrome’ [31]. The case described in our cohort further supports the notion that the phenotypic spectrum associated with GJA8 mutations extends beyond isolated cataract.

In the current study, a novel SOX2 heterozygous VUS (c.178G>C) was identified in a stillborn fetus (F12/P14) presenting with syndromic A/M. The recurrence of the condition in the family, despite both parents being clinically unaffected, raises the possibility of germline mosaicism. This phenomenon has previously been reported in association with SOX2 anophthalmia syndrome in four families where maternal mosaicism was identified [32-35]. To look for a potential mosaicism, we recommend that parents should undergo molecular testing of different tissues. Identifying mosaicism is important for accurately determining the recurrence risk and guiding genetic counseling.

Heterozygous pathogenic variants in CHD7 have been well documented in individuals diagnosed with CHARGE syndrome [36-39]. Within the current cohort, two patients with syndromic A/M met the clinical criteria of CHARGE syndrome. A heterozygous CHD7 pathogenic variant (c.5405–17G>A, p.?) was identified in one patient (F20/P23), while no causative variant was detected in the second (F27/P30). Prior studies have reported variable detection rates of CHD7 mutations among clinically diagnosed CHARGE patients [36,40,41]. These discrepancies may be attributed to differences in patient selection criteria, the sensitivity of molecular techniques employed, or the presence of pathogenic variants in noncoding regions of CHD7 or other critical regulatory elements not typically covered by standard ES.

Oculoauricular syndrome is a very rare AR genetic disorder, caused by biallelic LOF mutations in HMX1. Since its initial description by Schorderet et al. in 2008 [42], affected individuals from four families have been documented, all presenting with ocular anomalies and auricular malformations, while displaying normal neurodevelopment [42-45]. Here, we report a novel HMX1 variant (c.570_571delG, (p.E191Rfs*34) identified in a consanguineous Egyptian family, bringing the total number of reported families to five. Additional to the typical features of the syndrome, the patient (F21/P24) demonstrated psychomotor impairment and an abnormal gait – features not previously reported. The identification of the fifth HMX1 variant further supports the gene’s role in the pathogenesis of the syndrome and suggests a broader phenotypic variability than previously recognized.

In this study, one notable example of phenotypic expansions was the detection of a homozygous DPH1 variant (c.359T>C, p.Leu120Pro) associated with bilateral microphthalmia (F22/P25). Biallelic DPH1 variants have been recently linked to a rare neurodevelopmental disorder known as DEDSSH. To date, only 17 affected individuals from seven families have been reported, the majority were from Middle Eastern countries [46-51]. To the best of our knowledge, our case is the first reported from Egypt. The presence of severe microphthalmia in our patient suggests that ocular involvement may be an underrecognized feature of DPH1-related disorders. Further clinical, genetic, and functional studies involving larger cohorts are needed to confirm this potential association.

Moreover, this study further expands the molecular and phenotypic spectrum associated with ZBTB11 mutations by reporting a novel splicing variant (c.1623+2T>G) in an infant (F16/P18) presenting with bilateral microphthalmia. Biallelic variants in ZBTB11 have been linked to a neurodevelopmental condition (intellectual developmental disorder 69), characterized by ID, motor delay, and, in some cases, cataract [52]. Additional to the typical features of the syndrome, our patient exhibited bilateral microphthalmia and congenital heart disease – findings not commonly reported in association with ZBTB11 mutations. While most previously described variants have been missense mutations, the variant identified in this study is a predicted LOF mutation, which may explain the phenotypic expansion. Yet, the potential association of ZBTB11 mutations with ocular and cardiac anomalies warrants further investigation in larger cohorts. To the best of our knowledge, this is also the first documented case of ZBTB11-related disorder among Egyptians.

In the present study, we report a novel homozygous pathogenic nonsense variant in the B3GALNT2 gene in an infant (F17/P19) presenting with microphthalmia, congenital hydrocephalus and diffuse cobblestone-type II lissencephaly. This severe phenotype is consistent with previously reported cases involving truncating B3GALNT2 mutation, which are predicted to result in nonsense-mediated decay and are associated with severe clinical manifestations [53].

Despite the relatively high diagnostic yield, 48% of the tested families in this study remained without a definitive molecular diagnosis. This might suggest that additional genetic factors, such as mutations in yet undiscovered genes, may play a role in the etiology of A/M. It could also be attributed to the limitations inherent to the used methodology, which cannot detect non-coding, regulatory and variants in genes with corresponding pseudogenes, gene fusions, large and balanced chromosomal rearrangements, uniparental disomies, mosaicism and repeat expansions. Another consideration is that ES has a high likelihood of identifying VUSs. The identification of VUSs in two of our patients underscores the challenges in interpreting genomic data and highlights the need for complementary approaches, including functional studies and segregation analyses. Nonetheless, the main limitation was the use of proband-only ES in some cases, rather than the trio approach, due to financial constraints. For the unsolved families, we recommended further genetic testing using methylation profiling or genome sequencing (GS). Among the cohort, several families, including Family-12 with two similarly affected siblings, have already proceeded to GS, but the results are still pending.

Conclusion

The study provides a comprehensive molecular and clinical characterization of Egyptian patients with A/M. By reporting novel variants and unusual phenotypes, we contribute to expanding both the mutational landscape of A/M and the phenotypic spectrum of associated syndromes. Our results also support the genetic heterogeneity that underlies A/M etiology and reinforce the value of genetic testing for accurate diagnosis, counseling and future management. Nevertheless, further studies involving larger cohorts and a genome-wide approach are needed to uncover additional developmental genes.

Appendix 1. Supplementary Table 1.

Appendix 2. Supplementary Figure 1.

Appendix 3. Supplementary Table 2.

Appendix 4. Supplementary Figure 2.

Appendix 5. Supplemental Table 3.

Acknowledgments

We thank the patients and their families for their participation. Our thanks go to 3billion laboratory (Seoul, South Korea) for providing free ES to some patients participating in this study. Statement of Ethics: The legal guardians of all participants provided written informed consents, aligned with the guidelines of the Medical Research Institute Ethics Committee (IORG0008812). Specific written permission was obtained for the publication of photographs presented in this article. Conflict of interest: The authors declare no conflict of interest. Data availability statement: The data supporting the findings of this study are available upon request. Funding statement: The authors declare no financial directly or indirectly related to the work submitted for publication. Author contributions: GE: Recruiting the study cohort, conducting the clinical and cytogenetic work and preparing the first draft of the paper. AN, KN, DM, AA: All participated in the clinical and laboratory work and the analysis of data. MA, DS, NB, NE, EA: Providing the idea of this research, designing the study protocol, interpreting results, critically revising and final editing of the manuscript. All authors have read and approved the final manuscript.

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