Phenylalanine-associated ocular risk stratification in early-treated children with phenylketonuria: a cross-sectional study

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This study aimed to evaluate ocular findings in children with PKU and to examine their association with serum phenylalanine levels, with particular focus on quantitative risk stratification. Methods This cross-sectional study included 33 children less than 18 years diagnosed with phenylketonuria (PKU) by neonatal or selective screening and receiving regular follow-up at the Metabolic unit of Mansoura University Children’s Hospital, after obtaining informed parental consent. All participants underwent a comprehensive ophthalmological evaluation, including visual acuity assessment, cycloplegic refraction, anterior segment examination, fundus evaluation, intraocular pressure measurement, and ocular motility assessment. Serum phenylalanine levels were recorded, and receiver operating characteristic (ROC) curve analyses were performed to explore phenylalanine thresholds associated with ocular abnormalities. Results Ocular abnormalities were observed in a substantial proportion of participants and involved both anterior and posterior segments. Higher serum phenylalanine levels were significantly associated with increased likelihood of ocular involvement. ROC analysis identified phenylalanine ranges associated with elevated ocular risk, providing a quantitative framework for metabolic risk stratification.. Conclusion Ocular abnormalities may occur in early-treated children with PKU and appear to be associated with phenylalanine control. Quantitative phenylalanine thresholds identified through ROC analysis may support risk-stratified ophthalmic surveillance strategies in pediatric PKU. Phenylketonuria Ocular findings Diet regiment Figures Figure 1 Figure 2 Figure 3 What is already known PKU primarily affects the central nervous system, and elevated phenylalanine levels have been associated with visual dysfunction and occasional ocular abnormalities such as hypopigmentation, refractive errors, and cataract, mostly reported as isolated findings. What is new Ocular abnormalities are common and multisystemic in children with PKU and show a clear dose–response relationship with serum phenylalanine levels; this study defines phenylalanine cutoff values (>500 and >1000 µmol/L) associated with increased ocular risk, supporting the eye as a sensitive metabolic target and a potential tool for phenylalanine-guided ophthalmic screening. Introduction Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of the enzyme phenylalanine hydroxylase, resulting in elevated blood phenylalanine (Phe) concentrations. Before the introduction of neonatal screening programs in the 1960s, untreated PKU commonly led to severe intellectual disability, emotional and psychosocial disturbances, and irreversible neurological damage( 1 ). Since the implementation of national neonatal PKU screening programs, early diagnosis and dietary intervention have markedly improved neurological outcomes. In Egypt, a national neonatal PKU screening initiative was established in collaboration with the Ministry of Health in 2015, enabling early identification of affected infants and initiation of a Phe-restricted diet, which effectively lowers blood phenylalanine levels and prevents severe intellectual disability( 2 ). Current recommendations emphasize lifelong adherence to dietary treatment in individuals with PKU. However, maintaining strict dietary control becomes increasingly challenging during late childhood and adolescence, when social factors and dietary independence often result in suboptimal metabolic control( 3 ). Regular monitoring of blood phenylalanine concentrations remains the cornerstone of treatment follow-up, and specific target ranges have been recommended for special populations, including women planning pregnancy and during gestation (120–360 µmol/L) ( 4 ). PKU is conventionally classified according to pretreatment blood phenylalanine concentrations and dietary phenylalanine tolerance, ranging from classical PKU (cPKU), with pretreatment Phe levels exceeding 1200 µmol/L, to mild PKU (mPKU; 600–1200 µmol/L) and mild hyperphenylalaninemia (MHP; 120–600 µmol/L) ( 5 ). At elevated concentrations, phenylalanine competes with other large neutral amino acids for transport across the blood–brain barrier and into peripheral tissues, leading to amino acid imbalances that contribute to impaired myelination and oligodendrocyte dysfunction ( 6 , 7 ). The clinical and metabolic phenotype of PKU is highly heterogeneous and largely determined by residual phenylalanine hydroxylase activity. Lower residual enzyme activity is typically associated with higher blood phenylalanine levels and more severe manifestations in the absence of treatment( 8 ). Beyond its well-established neurological effects, increasing evidence suggests that chronic hyperphenylalaninemia may also affect ocular structures and visual function. Dopamine, synthesized from phenylalanine-derived tyrosine, plays a key role in retinal signaling and regulation of eye growth. Altered dopaminergic pathways have been implicated in the development of myopia and visual dysfunction in experimental models of PKU, providing a plausible mechanistic link between metabolic imbalance and ocular involvement ( 9 ). Nevertheless, available data on ocular abnormalities in PKU—including iris and retinal hypopigmentation, cataract formation, corneal opacities, and functional visual impairment—remain limited and fragmented, particularly in pediatric populations receiving early dietary treatment ( 10 ) . Accordingly, the present study aimed to evaluate ocular findings in children with early-treated PKU diagnosed through neonatal or selective screening and to examine the association between ophthalmological manifestations and serum phenylalanine levels in this population. Patients and Methods This cross-sectional study included 33 pediatric patients diagnosed with phenylketonuria (PKU) through neonatal or selective screening who were attending the Genetics and Metabolic Outpatient Clinic at Mansoura University Children’s Hospital (MUCH). Written informed consent was obtained from the parents or legal guardians of all participants prior to enrollment. Eligible participants were children aged 3–18 years with a confirmed diagnosis of PKU who were under regular follow-up at the metabolic unit of MUCH. Newborn screening was performed using dried blood spot samples collected on Whatman® 903 filter paper from heel-stick blood between the third and seventh days of life. Samples were processed according to the NeoBase™ non-derivatized tandem mass spectrometry (MS/MS) kit protocol and analyzed using the TQD MS/MS system. The screening panel included phenylalanine, tyrosine, and acylcarnitines. Positive screening results were confirmed by plasma amino acid analysis using chromatography to quantify phenylalanine levels ( 11 ). Exclusion Criteria: Children aged below 3 or above 18 years. History of systemic disease known to affect the retina e.g., diabetes. History of other ocular disease, previous ocular trauma or operation. History of any neurological disease unrelated to PKU. Prematurity < 36 weeks of gestational age. Interfering medical treatment. Ophthalmological examination All participants underwent comprehensive ophthalmological evaluation at the Mansoura Ophthalmology Center, Mansoura University. Examinations included: Measurement of visual acuity using age-appropriate Snellen charts, with values converted to logarithm of the minimum angle of resolution (logMAR) units for statistical analysis Refractive assessment when indicated, based on objective cycloplegic refraction using a conventional autorefractometer Measurement of intraocular pressure using a non-contact tonometer Detailed anterior segment examination using slit-lamp biomicroscopy Dilated fundus examination using slit-lamp biomicroscopy with direct and indirect ophthalmoscopy Assessment of ocular motility Ethical consideration The study was approved by Institutional Review Board, Mansoura Faculty of Medicine, Mansoura University. Code Number:R.23.12.2432 Statistical analysis: Mean and standard deviation of all parameters in serum will be calculated. Student t-test will be used for comparison of means and p < 0.05 will be considered significant for parametric data. Wilcoxon test will be used for non-parametric data, And p < 0.05 will be considered significant. Descriptive statistics were used to analyze demographic data and phenylalanine levels. The relationship between phenylalanine levels and ocular findings was assessed using correlation analysis. Prognostic value was determined using receiver operating characteristic (ROC) curve analysis. Results 1. Demographic and Biochemical Characteristics A total of 33 children with phenylketonuria (PKU) were included. The cohort consisted of 13 males (39%) and 20 females (61%), with a female-to-male ratio of 1.5:1 (p = 0.50). The mean age was 5.6 ± 2.1 years. Median serum phenylalanine (Phe) level was 433 µmol/L (range 110–1250 µmol/L). 2. General Ophthalmic Findings Ophthalmological abnormalities involving both anterior and posterior segments were identified in a proportion of the studied patients. Corneal clouding was observed in 5 children (15.2%), iris hypopigmentation in 4 (12.1%), retinal hypopigmentation in 11 (33.3%), and posterior polar cataract in 3 patients (9.1%). Optic disc abnormalities, including disc pallor or myopic disc appearance, were detected in 5 children (15.2%). Refractive errors were identified in 8 patients (24.2%), including myopia (n = 5) and astigmatism (n = 3). Ocular motility abnormalities (eso- or exotropia) were present in 4 patients (12.1%). Intraocular pressure measurements were within normal limits in all examined children (Table 1 and Fig. 1 ). Rare findings such as vitreous floaters and retinal vascular tortuosity were observed in single cases and were therefore interpreted descriptively. Table 1 General ocular findings among PKU patients Ocular Finding Normal n (%) Abnormal n (%) Abnormality Type p-value Cornea 28 (84.8%) 5 (15.2%) Clouding 0.003 Iris 29 (87.9%) 4 (12.1%) Hypopigmentation 0.001 Vitreous 32 (97%) 1 (3%) Floaters ------* Retina 22 (66.7%) 11 (33.3%) Hypopigmentation 0.273 Retinal Vessels 32 (97%) 1 (3%) Tortuosity ------* Lens 30 (90.9%) 3 (9.1%) Posterior polar cataract < 0.001 Optic Disc 28 (84.8%) 5 (15.2%) Disc pallor or myopic disc 0.003 Refractive Error 25 (75.8%) 8 (24.2%) Myopia (n = 5), Astigmatism (n = 3) 0.047 Ocular Motility 29 (87.9%) 4 (12.1%) Eso-/Exotropia < 0.001 IOP 33 (100%) 0 — — * interpreted descriptively as number of abnormalities in one group were less than 2 cases 3. Relationship Between Phenylalanine Levels and Specific Ocular Findings Median Phe concentrations were compared between normal and abnormal Median serum phenylalanine concentrations were compared between children with normal and abnormal findings for each ocular structure (Table 2 ). Higher median Phe levels were observed among patients with corneal clouding, cataract, retinal hypopigmentation, optic disc abnormalities, and refractive errors compared with those without these findings. The differences were statistically significant for corneal involvement (p = 0.026), cataract (p = 0.006), retinal hypopigmentation (p < 0.001), optic disc abnormalities (p = 0.005), and refractive errors (p = 0.040). Associations involving very infrequent abnormalities were not statistically emphasized due to limited event numbers. Table 2 Specific ocular findings and phenylalanine relationship Ocular Structure Normal Phe (µmol/L) Abnormal Phe (µmol/L) p-value Cornea 208.5 (110–1250) 1020 (530–1145) 0.026 Iris 216 (110–1250) 1010 (637–1040) 0.80 Lens (Cataract) 302 (110–1200) 1145 (1040–1250) 0.006 Retina 195 (110–1145) 1020 (523–1250) < 0.001 Retinal Vessels 411 (110–1250) 1200 0.121 Vitreous 411 (110–1200) 1250 0.061 Refractive Error 200 (110–1200) 1020 (201–1250) 0.040 Optic Disc 208 (110–1250) 1090 (600–1180) 0.005 Figure 2 . Median serum phenylalanine levels in children with normal versus abnormal ocular findings. Across corneal, lenticular, retinal, and optic disc assessments, higher phenylalanine concentrations were observed in participants with abnormal findings. Values are presented as median µmol/L. 4. Predictive Value of Phenylalanine for Ocular Pathology (ROC Analysis) Receiver operating characteristic (ROC) curve analysis was performed to explore the discriminative ability of serum phenylalanine levels for selected ocular abnormalities ( Table 3 and Fig. 3). Phenylalanine demonstrated moderate to high discriminative performance for corneal clouding (AUC = 0.81), retinal hypopigmentation (AUC = 0.88), and optic disc abnormalities (AUC = 0.88), and high discriminative performance for cataract (AUC = 0.94). Cutoff values around 500 µmol/L were associated with increased probability of corneal and retinal abnormalities, while values exceeding 1000 µmol/L were associated with lens and optic disc involvement. Given the sample size, these thresholds should be interpreted as exploratory. Table 3 ROC Performance of Phenylalanine for Predicting Ocular Abnormalities Ocular Outcome AUC 95% CI p-value Optimal Cutoff (µmol/L) Sensitivity 1–Specificity Corneal Clouding 0.811 0.666–0.956 0.029 501 100% 36% Iris Hypopigmentation 0.784 0.636–0.932 0.069 501 100% 38% Cataract 0.944 0.861–1.000 0.012 1020 100% 13.3% Retinal Hypopigmentation 0.884 0.883–1.000 < 0.001 501 100% 18% Optic Disc Abnormality 0.879 0.753–1.000 0.008 1020 80% 10.7% Figure 3. Receiver operating characteristic (ROC) curves illustrating the discriminative performance of serum phenylalanine levels for selected ocular abnormalities in children with phenylketonuria. Curves are shown for corneal clouding, cataract, retinal hypopigmentation, and iris hypopigmentation. The area under the curve (AUC) reflects the ability of phenylalanine levels to distinguish between children with and without each ocular finding. The diagonal line represents no discriminative performance (AUC = 0.5). 5. Correlation Analysis Higher serum phenylalanine levels were significantly correlated with worse visual acuity, expressed as higher logMAR values (Spearman r = 0.843, p < 0.001). Correlations between phenylalanine levels and axial length or keratometric parameters (K1 and K2) did not reach statistical significance. Parameter Correlation (Spearman r) p-value Visual Acuity 0.843 < 0.001 K1 0.400 0.505 K2 0.400 0.505 Axial Length 0.700 0.188 Discussion The present study demonstrates that ocular abnormalities are relatively common among children with phenylketonuria (PKU), and many of these findings—including corneal clouding, iris and retinal hypopigmentation, posterior polar cataract, optic disc abnormalities, refractive errors, and strabismus—show strong associations with elevated serum phenylalanine (Phe) levels. Recent literature increasingly recognizes the eye as an early metabolic sensor in PKU, paralleling the CNS in its susceptibility to phenylalanine toxicity ( 9 , 12 ). Our findings are consistent with this evolving understanding and further expand the evidence linking metabolic control to ocular integrity. Pigmentary changes were particularly prominent in children with higher Phe levels. Modern mechanistic studies show that excess phenylalanine disrupts melanin biosynthesis by reducing tyrosine availability and directly inhibiting tyrosinase and related melanogenic enzymes( 13 , 14 ). Clinical observations in contemporary PKU cohorts confirm that iris and retinal hypopigmentation remain more common in individuals with suboptimal metabolic control( 9 ) .The strong relationship between Phe and hypopigmentation in our dataset supports these biochemical and clinical findings and highlights melanin pathways as a sensitive metabolic target. Retinal and optic nerve abnormalities observed in our cohort are also strongly supported by recent imaging and functional studies. High-resolution OCT studies from the past five years report significant thinning of the peripapillary retinal nerve fiber layer (RNFL), reduced ganglion cell–inner plexiform layer (GCIPL), and microstructural neuroretinal changes in PKU patients—particularly in those with elevated lifetime or current Phe levels( 15 – 18 ). Functional studies show reduced contrast sensitivity, delayed visual processing, and abnormal visual evoked potentials in poorly controlled patients ( 10 , 19 ). Our findings of retinal hypopigmentation, optic disc pallor, and a strong correlation between Phe and reduced visual acuity closely align with this recent evidence, suggesting that ocular neurodegeneration in PKU is both structural and functional in nature. Lens involvement also showed a clear link to metabolic control. While cataract in PKU has traditionally been described in older literature( 13 ), recent reports continue to note lens opacities in children with severe or fluctuating hyperphenylalaninemia( 20 ). Oxidative stress from phenylalanine derivatives and metabolic lens vulnerability remains the leading hypotheses( 21 ). In our cohort, cataract occurred exclusively in children with very high Phe levels, and phenylalanine demonstrated excellent predictive discrimination (AUC 0.94), providing one of the most robust quantitative validations of this association. Refractive errors and strabismus were also significantly associated with higher Phe levels in our patients. Recent developmental-visual studies indicate that abnormal retinal signaling, impaired dopamine pathways, and altered visual cortex maturation in PKU may contribute to refractive instability and ocular misalignment( 22 – 24 ) .Our results are consistent with this work and indicate that mild-to-moderate metabolic dysregulation during critical periods of visual development may be sufficient to disrupt emmetropization and ocular motor control. One of the most important contributions of this study is the derivation of phenylalanine cutoff values associated with multiple ocular abnormalities using ROC analysis. To our knowledge, no recent PKU studies have quantified specific phenylalanine thresholds for corneal, lenticular, retinal, and optic nerve changes within a single cohort. Our data support a multi-tiered model whereby ocular risk rises significantly above 500 µmol/L and becomes high above 1000 µmol/L—information that has not been provided in previous ophthalmic or metabolic studies. Together, our findings reveal a consistent, multi-tissue dose–response pattern: as phenylalanine levels rise, abnormalities appear progressively across corneal, pigmentary, lenticular, retinal, and optic nerve structures. Earlier studies examined each structure in isolation, but our integrated dataset demonstrates that the entire eye behaves as a metabolically sensitive organ. This unified pattern supports the development of ophthalmic screening strategies tailored to phenylalanine levels and may assist clinicians in identifying patients who require more frequent ocular monitoring. In summary, this study confirms that ocular involvement in PKU is metabolically driven, clinically meaningful, and detectable even in childhood. Elevated phenylalanine was consistently linked to structural abnormalities and functional visual loss. By integrating detailed ophthalmic evaluation with quantitative ROC-based phenylalanine thresholds, our study introduces novel evidence that may improve targeted ophthalmic surveillance and support more stringent metabolic management to prevent long-term visual morbidity in children with PKU. Conclusion Ocular abnormalities are common and multisystemic in children with phenylketonuria and are associated with increasing serum phenylalanine levels. Exploratory analyses suggest that phenylalanine concentrations above approximately 500 µmol/L are associated with an increased likelihood of ocular involvement, with more severe abnormalities observed at levels exceeding 1000 µmol/L. These findings support the eye as a metabolically sensitive organ and highlight the potential value of phenylalanine-guided ophthalmic surveillance in pediatric PKU. STUDY LIMITATION and FUTURE DIRECTIONS This study’s cross-sectional design and single-center cohort limit causal inference and generalizability. Visual assessments were sometimes challenging in younger children, and detailed imaging was not available for all participants. Longitudinal studies are needed to track the progression of ocular changes in PKU and evaluate the impact of early and sustained metabolic control. Advanced imaging and functional assessments could further clarify structure–function relationships, while multicenter studies may validate phenylalanine thresholds for risk stratification and guide personalized ophthalmic screening protocols Abbreviations PKU Phenylketonuria Phe Phenylalanine ROC Receiver Operating Characteristic AUC Area Under the Curve IOP Intraocular Pressure logMAR Logarithm of the Minimum Angle of Resolution MS/MS Tandem Mass Spectrometry PAH Phenylalanine Hydroxylase MHP Mild Hyperphenylalaninemia cPKU Classical Phenylketonuria mPKU Mild Phenylketonuria MUCH Mansoura University Children’s Hospital MOC Mansoura Ophthalmology Center Declarations Ethical approval :This study was conducted by the principles outlined in the Declaration of Helsinki. The study protocol was approved by the Research Ethics Committee of Faculty of Medicine, Mansoura University (Code Number :R.23.12.2432) and followed by Institutional Research Board. Informed consents were obtained from all parents of the children included in this study. Consent to participate : informed consent was obtained from all patient caregivers before inclusion in the study. Clinical trial number : Not applicable Consent for publication : Not Applicable Data availability : The datasets generated and/or analyzed during the current study are not publicly available due to institutional and ethical restrictions but are available from the corresponding author on reasonable request Conflict of interest :The authors declare that no conflicts of interest associated with this manuscript. Research funding : the authors declare that no funds ,grants, or other support were received during the preparation of this research . Author contributions : H.E. contributed to patient recruitment, clinical metabolic assessment, data collection, and drafted the initial manuscript. D.L. performed comprehensive ophthalmological examinations and contributed to data acquisition and interpretation. M.A.H. conceptualized and designed the study, performed statistical analysis, supervised the research process, contributed to data interpretation, and critically revised the manuscript for important intellectual content. All authors approved the final manuscript and agreed to be accountable for all aspects of the work Acknowledgments : The authors would like to thank the children and their families for their participation in this study. We also acknowledge the nursing staff and laboratory personnel at Mansoura University Children’s Hospital and Mansoura ophthalmology center for their assistance in patient coordination and biochemical assessments. References Waisbren SE, Noel K, Fahrbach K, Cella C, Frame D, Dorenbaum A, et al. Phenylalanine blood levels and clinical outcomes in phenylketonuria: a systematic literature review and meta-analysis. 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Cell Mol Neurobiol. 2011;31(5):653–62. Zhou X, Pardue MT, Iuvone PM, Qu J. Dopamine signaling and myopia development: What are the key challenges. Prog Retin Eye Res. 2017;61:60–71. Tian T, Zou L, Wang S, Liu R, Liu H. The Role of Dopamine in Emmetropization Modulated by Wavelength and Temporal Frequency in Guinea Pigs. Invest Ophthalmol Vis Sci. 2021;62(12):20. Bergen MA, Park HN, Chakraborty R, Landis EG, Sidhu C, He L, et al. Altered Refractive Development in Mice With Reduced Levels of Retinal Dopamine. Invest Ophthalmol Vis Sci. 2016;57(10):4412–9. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8929509","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611565131,"identity":"431c323e-3ab5-44e0-b9b7-1c85e5bb76cd","order_by":0,"name":"Heba ElTaher","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Heba","middleName":"","lastName":"ElTaher","suffix":""},{"id":611565132,"identity":"870cf2ad-7364-4790-b74d-883ec36c1718","order_by":1,"name":"Dina Laimon","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Dina","middleName":"","lastName":"Laimon","suffix":""},{"id":611565133,"identity":"f035306b-3b7a-4c6b-85c8-d14aad82427b","order_by":2,"name":"Mohamed Abdelghafar Hussein","email":"data:image/png;base64,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","orcid":"","institution":"Kafrelsheikh University","correspondingAuthor":true,"prefix":"","firstName":"Mohamed","middleName":"Abdelghafar","lastName":"Hussein","suffix":""}],"badges":[],"createdAt":"2026-02-20 23:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8929509/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8929509/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105497918,"identity":"7e16ca90-f73c-4ed2-b5c0-b830a46b35aa","added_by":"auto","created_at":"2026-03-26 16:52:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":381678,"visible":true,"origin":"","legend":"\u003cp\u003ePrevalence of ocular abnormalities in children with phenylketonuria. Retinal hypopigmentation and refractive errors were the most frequent findings\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8929509/v1/88277078dbf8c2c904d2f444.png"},{"id":105497916,"identity":"6d16b449-7d7a-40a2-99e0-708123afa561","added_by":"auto","created_at":"2026-03-26 16:52:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61331,"visible":true,"origin":"","legend":"\u003cp\u003eMedian phenylalanine levels by ocular status\u003c/p\u003e\n\u003cp\u003eMedian serum phenylalanine levels in children with normal versus abnormal ocular findings. Across corneal, lenticular, retinal, and optic disc assessments, higher phenylalanine concentrations were observed in participants with abnormal findings. Values are presented as median µmol/L.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8929509/v1/8bdf6193d36b7bd1143aaa58.png"},{"id":105497917,"identity":"27682ea8-26a8-46cf-ae3d-d078983bf1bd","added_by":"auto","created_at":"2026-03-26 16:52:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185907,"visible":true,"origin":"","legend":"\u003cp\u003eReceiver operating characteristic (ROC) curves illustrating the discriminative performance of serum phenylalanine levels for selected ocular abnormalities in children with phenylketonuria. Curves are shown for corneal clouding, cataract, retinal hypopigmentation, and iris hypopigmentation. The area under the curve (AUC) reflects the ability of phenylalanine levels to distinguish between children with and without each ocular finding. The diagonal line represents no discriminative performance (AUC = 0.5).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8929509/v1/1202cf0e124759f8ffdc2bca.png"},{"id":105888667,"identity":"e72c854e-0fcb-4d18-90de-110fab6af82c","added_by":"auto","created_at":"2026-04-01 07:44:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1291032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8929509/v1/35390908-1df8-43e9-9012-d8e0c47ef2bc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phenylalanine-associated ocular risk stratification in early-treated children with phenylketonuria: a cross-sectional study","fulltext":[{"header":"What is already known","content":"\u003cp\u003ePKU primarily affects the central nervous system, and elevated phenylalanine levels have been associated with visual dysfunction and occasional ocular abnormalities such as hypopigmentation, refractive errors, and cataract, mostly reported as isolated findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhat is new\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOcular abnormalities are common and multisystemic in children with PKU and show a clear dose\u0026ndash;response relationship with serum phenylalanine levels; this study defines phenylalanine cutoff values (\u0026gt;500 and \u0026gt;1000 \u0026micro;mol/L) associated with increased ocular risk, supporting the eye as a sensitive metabolic target and a potential tool for phenylalanine-guided ophthalmic screening.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePhenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of the enzyme phenylalanine hydroxylase, resulting in elevated blood phenylalanine (Phe) concentrations. Before the introduction of neonatal screening programs in the 1960s, untreated PKU commonly led to severe intellectual disability, emotional and psychosocial disturbances, and irreversible neurological damage(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Since the implementation of national neonatal PKU screening programs, early diagnosis and dietary intervention have markedly improved neurological outcomes. In Egypt, a national neonatal PKU screening initiative was established in collaboration with the Ministry of Health in 2015, enabling early identification of affected infants and initiation of a Phe-restricted diet, which effectively lowers blood phenylalanine levels and prevents severe intellectual disability(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Current recommendations emphasize lifelong adherence to dietary treatment in individuals with PKU. However, maintaining strict dietary control becomes increasingly challenging during late childhood and adolescence, when social factors and dietary independence often result in suboptimal metabolic control(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Regular monitoring of blood phenylalanine concentrations remains the cornerstone of treatment follow-up, and specific target ranges have been recommended for special populations, including women planning pregnancy and during gestation (120\u0026ndash;360 \u0026micro;mol/L) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePKU is conventionally classified according to pretreatment blood phenylalanine concentrations and dietary phenylalanine tolerance, ranging from classical PKU (cPKU), with pretreatment Phe levels exceeding 1200 \u0026micro;mol/L, to mild PKU (mPKU; 600\u0026ndash;1200 \u0026micro;mol/L) and mild hyperphenylalaninemia (MHP; 120\u0026ndash;600 \u0026micro;mol/L) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). At elevated concentrations, phenylalanine competes with other large neutral amino acids for transport across the blood\u0026ndash;brain barrier and into peripheral tissues, leading to amino acid imbalances that contribute to impaired myelination and oligodendrocyte dysfunction (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe clinical and metabolic phenotype of PKU is highly heterogeneous and largely determined by residual phenylalanine hydroxylase activity. Lower residual enzyme activity is typically associated with higher blood phenylalanine levels and more severe manifestations in the absence of treatment(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond its well-established neurological effects, increasing evidence suggests that chronic hyperphenylalaninemia may also affect ocular structures and visual function. Dopamine, synthesized from phenylalanine-derived tyrosine, plays a key role in retinal signaling and regulation of eye growth. Altered dopaminergic pathways have been implicated in the development of myopia and visual dysfunction in experimental models of PKU, providing a plausible mechanistic link between metabolic imbalance and ocular involvement (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Nevertheless, available data on ocular abnormalities in PKU\u0026mdash;including iris and retinal hypopigmentation, cataract formation, corneal opacities, and functional visual impairment\u0026mdash;remain limited and fragmented, particularly in pediatric populations receiving early dietary treatment (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eAccordingly, the present study aimed to evaluate ocular findings in children with early-treated PKU diagnosed through neonatal or selective screening and to examine the association between ophthalmological manifestations and serum phenylalanine levels in this population.\u003c/p\u003e"},{"header":"Patients and Methods","content":"\u003cp\u003eThis cross-sectional study included 33 pediatric patients diagnosed with phenylketonuria (PKU) through neonatal or selective screening who were attending the Genetics and Metabolic Outpatient Clinic at Mansoura University Children\u0026rsquo;s Hospital (MUCH). Written informed consent was obtained from the parents or legal guardians of all participants prior to enrollment.\u003c/p\u003e \u003cp\u003eEligible participants were children aged 3\u0026ndash;18 years with a confirmed diagnosis of PKU who were under regular follow-up at the metabolic unit of MUCH. Newborn screening was performed using dried blood spot samples collected on Whatman\u0026reg; 903 filter paper from heel-stick blood between the third and seventh days of life. Samples were processed according to the NeoBase\u0026trade; non-derivatized tandem mass spectrometry (MS/MS) kit protocol and analyzed using the TQD MS/MS system. The screening panel included phenylalanine, tyrosine, and acylcarnitines. Positive screening results were confirmed by plasma amino acid analysis using chromatography to quantify phenylalanine levels (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExclusion Criteria:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eChildren aged below 3 or above 18 years.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eHistory of systemic disease known to affect the retina e.g., diabetes.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eHistory of other ocular disease, previous ocular trauma or operation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eHistory of any neurological disease unrelated to PKU.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePrematurity\u0026thinsp;\u0026lt;\u0026thinsp;36 weeks of gestational age.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eInterfering medical treatment.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOphthalmological examination\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e All participants underwent comprehensive ophthalmological evaluation at the Mansoura Ophthalmology Center, Mansoura University. Examinations included:\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eMeasurement of visual acuity using age-appropriate Snellen charts, with values converted to logarithm of the minimum angle of resolution (logMAR) units for statistical analysis\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eRefractive assessment when indicated, based on objective cycloplegic refraction using a conventional autorefractometer\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMeasurement of intraocular pressure using a non-contact tonometer\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eDetailed anterior segment examination using slit-lamp biomicroscopy\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eDilated fundus examination using slit-lamp biomicroscopy with direct and indirect ophthalmoscopy\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eAssessment of ocular motility\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthical consideration\u003c/h3\u003e\n\u003cp\u003e The study was approved by Institutional Review Board, Mansoura Faculty of Medicine, Mansoura University. Code Number:R.23.12.2432\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eMean and standard deviation of all parameters in serum will be calculated. Student t-test will be used for comparison of means and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 will be considered significant for parametric data. Wilcoxon test will be used for non-parametric data,\u003c/p\u003e \u003cp\u003eAnd p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 will be considered significant. Descriptive statistics were used to analyze demographic data and phenylalanine levels. The relationship between phenylalanine levels and ocular findings was assessed using correlation analysis. Prognostic value was determined using receiver operating characteristic (ROC) curve analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e1. Demographic and Biochemical Characteristics\u003c/p\u003e \u003cp\u003eA total of 33 children with phenylketonuria (PKU) were included.\u003c/p\u003e \u003cp\u003eThe cohort consisted of 13 males (39%) and 20 females (61%), with a female-to-male ratio of 1.5:1 (p\u0026thinsp;=\u0026thinsp;0.50). The mean age was 5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 years. Median serum phenylalanine (Phe) level was 433 \u0026micro;mol/L (range 110\u0026ndash;1250 \u0026micro;mol/L).\u003c/p\u003e \u003cp\u003e2. General Ophthalmic Findings\u003c/p\u003e \u003cp\u003eOphthalmological abnormalities involving both anterior and posterior segments were identified in a proportion of the studied patients. Corneal clouding was observed in 5 children (15.2%), iris hypopigmentation in 4 (12.1%), retinal hypopigmentation in 11 (33.3%), and posterior polar cataract in 3 patients (9.1%). Optic disc abnormalities, including disc pallor or myopic disc appearance, were detected in 5 children (15.2%).\u003c/p\u003e \u003cp\u003eRefractive errors were identified in 8 patients (24.2%), including myopia (n\u0026thinsp;=\u0026thinsp;5) and astigmatism (n\u0026thinsp;=\u0026thinsp;3). Ocular motility abnormalities (eso- or exotropia) were present in 4 patients (12.1%). Intraocular pressure measurements were within normal limits in all examined children (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRare findings such as vitreous floaters and retinal vascular tortuosity were observed in single cases and were therefore interpreted descriptively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGeneral ocular findings among PKU patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOcular Finding\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbnormal n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbnormality Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCornea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28 (84.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (15.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClouding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIris\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29 (87.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (12.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypopigmentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitreous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (97%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFloaters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e------*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypopigmentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.273\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetinal Vessels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (97%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTortuosity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e------*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 (90.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (9.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePosterior polar cataract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptic Disc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28 (84.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (15.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDisc pallor or myopic disc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRefractive Error\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 (75.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (24.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMyopia (n\u0026thinsp;=\u0026thinsp;5), Astigmatism (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.047\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOcular Motility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29 (87.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (12.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEso-/Exotropia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* interpreted descriptively as number of abnormalities in one group were less than 2 cases\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3. Relationship Between Phenylalanine Levels and Specific Ocular Findings\u003c/p\u003e \u003cp\u003eMedian Phe concentrations were compared between normal and abnormal Median serum phenylalanine concentrations were compared between children with normal and abnormal findings for each ocular structure (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Higher median Phe levels were observed among patients with corneal clouding, cataract, retinal hypopigmentation, optic disc abnormalities, and refractive errors compared with those without these findings. The differences were statistically significant for corneal involvement (p\u0026thinsp;=\u0026thinsp;0.026), cataract (p\u0026thinsp;=\u0026thinsp;0.006), retinal hypopigmentation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), optic disc abnormalities (p\u0026thinsp;=\u0026thinsp;0.005), and refractive errors (p\u0026thinsp;=\u0026thinsp;0.040). Associations involving very infrequent abnormalities were not statistically emphasized due to limited event numbers.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecific ocular findings and phenylalanine relationship\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOcular Structure\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal Phe (\u0026micro;mol/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbnormal Phe (\u0026micro;mol/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCornea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e208.5 (110\u0026ndash;1250)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1020 (530\u0026ndash;1145)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIris\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e216 (110\u0026ndash;1250)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1010 (637\u0026ndash;1040)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLens (Cataract)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e302 (110\u0026ndash;1200)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1145 (1040\u0026ndash;1250)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e195 (110\u0026ndash;1145)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1020 (523\u0026ndash;1250)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetinal Vessels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e411 (110\u0026ndash;1250)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitreous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e411 (110\u0026ndash;1200)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRefractive Error\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 (110\u0026ndash;1200)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1020 (201\u0026ndash;1250)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.040\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptic Disc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e208 (110\u0026ndash;1250)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1090 (600\u0026ndash;1180)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Median serum phenylalanine levels in children with normal versus abnormal ocular findings. Across corneal, lenticular, retinal, and optic disc assessments, higher phenylalanine concentrations were observed in participants with abnormal findings. Values are presented as median \u0026micro;mol/L.\u003c/p\u003e \u003cp\u003e4. Predictive Value of Phenylalanine for Ocular Pathology (ROC Analysis)\u003c/p\u003e \u003cp\u003eReceiver operating characteristic (ROC) curve analysis was performed to explore the discriminative ability of serum phenylalanine levels for selected ocular abnormalities \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003eand Fig.\u0026nbsp;3).\u003c/b\u003e Phenylalanine demonstrated moderate to high discriminative performance for corneal clouding (AUC\u0026thinsp;=\u0026thinsp;0.81), retinal hypopigmentation (AUC\u0026thinsp;=\u0026thinsp;0.88), and optic disc abnormalities (AUC\u0026thinsp;=\u0026thinsp;0.88), and high discriminative performance for cataract (AUC\u0026thinsp;=\u0026thinsp;0.94).\u003c/p\u003e \u003cp\u003eCutoff values around 500 \u0026micro;mol/L were associated with increased probability of corneal and retinal abnormalities, while values exceeding 1000 \u0026micro;mol/L were associated with lens and optic disc involvement. Given the sample size, these thresholds should be interpreted as exploratory.\u003c/p\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eROC Performance of Phenylalanine for Predicting Ocular Abnormalities\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eOcular Outcome\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAUC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eOptimal Cutoff (\u0026micro;mol/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003eSensitivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e1\u0026ndash;Specificity\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCorneal Clouding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.811\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0.666\u0026ndash;0.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e36%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eIris Hypopigmentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0.636\u0026ndash;0.932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e38%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCataract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0.861\u0026ndash;1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e1020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e13.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eRetinal Hypopigmentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.884\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0.883\u0026ndash;1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e18%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eOptic Disc Abnormality\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.879\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0.753\u0026ndash;1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e1020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e80%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e10.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3.\u003c/strong\u003e Receiver operating characteristic (ROC) curves illustrating the discriminative performance of serum phenylalanine levels for selected ocular abnormalities in children with phenylketonuria. Curves are shown for corneal clouding, cataract, retinal hypopigmentation, and iris hypopigmentation. The area under the curve (AUC) reflects the ability of phenylalanine levels to distinguish between children with and without each ocular finding. The diagonal line represents no discriminative performance (AUC\u0026thinsp;=\u0026thinsp;0.5).\u003c/p\u003e\n\u003cp\u003e5. Correlation Analysis\u003c/p\u003e\n\u003cp\u003eHigher serum phenylalanine levels were significantly correlated with worse visual acuity, expressed as higher logMAR values (Spearman r\u0026thinsp;=\u0026thinsp;0.843, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Correlations between phenylalanine levels and axial length or keratometric parameters (K1 and K2) did not reach statistical significance.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCorrelation (Spearman r)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eVisual Acuity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0.505\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eK2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0.505\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eAxial Length\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e0.700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0.188\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"Discussion","content":"\u003cp\u003e The present study demonstrates that ocular abnormalities are relatively common among children with phenylketonuria (PKU), and many of these findings\u0026mdash;including corneal clouding, iris and retinal hypopigmentation, posterior polar cataract, optic disc abnormalities, refractive errors, and strabismus\u0026mdash;show strong associations with elevated serum phenylalanine (Phe) levels. Recent literature increasingly recognizes the eye as an early metabolic sensor in PKU, paralleling the CNS in its susceptibility to phenylalanine toxicity (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Our findings are consistent with this evolving understanding and further expand the evidence linking metabolic control to ocular integrity.\u003c/p\u003e \u003cp\u003ePigmentary changes were particularly prominent in children with higher Phe levels. Modern mechanistic studies show that excess phenylalanine disrupts melanin biosynthesis by reducing tyrosine availability and directly inhibiting tyrosinase and related melanogenic enzymes(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Clinical observations in contemporary PKU cohorts confirm that iris and retinal hypopigmentation remain more common in individuals with suboptimal metabolic control(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) .The strong relationship between Phe and hypopigmentation in our dataset supports these biochemical and clinical findings and highlights melanin pathways as a sensitive metabolic target.\u003c/p\u003e \u003cp\u003eRetinal and optic nerve abnormalities observed in our cohort are also strongly supported by recent imaging and functional studies. High-resolution OCT studies from the past five years report significant thinning of the peripapillary retinal nerve fiber layer (RNFL), reduced ganglion cell\u0026ndash;inner plexiform layer (GCIPL), and microstructural neuroretinal changes in PKU patients\u0026mdash;particularly in those with elevated lifetime or current Phe levels(\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Functional studies show reduced contrast sensitivity, delayed visual processing, and abnormal visual evoked potentials in poorly controlled patients (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Our findings of retinal hypopigmentation, optic disc pallor, and a strong correlation between Phe and reduced visual acuity closely align with this recent evidence, suggesting that ocular neurodegeneration in PKU is both structural and functional in nature.\u003c/p\u003e \u003cp\u003eLens involvement also showed a clear link to metabolic control. While cataract in PKU has traditionally been described in older literature(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), recent reports continue to note lens opacities in children with severe or fluctuating hyperphenylalaninemia(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Oxidative stress from phenylalanine derivatives and metabolic lens vulnerability remains the leading hypotheses(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In our cohort, cataract occurred exclusively in children with very high Phe levels, and phenylalanine demonstrated excellent predictive discrimination (AUC 0.94), providing one of the most robust quantitative validations of this association.\u003c/p\u003e \u003cp\u003eRefractive errors and strabismus were also significantly associated with higher Phe levels in our patients. Recent developmental-visual studies indicate that abnormal retinal signaling, impaired dopamine pathways, and altered visual cortex maturation in PKU may contribute to refractive instability and ocular misalignment(\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) .Our results are consistent with this work and indicate that mild-to-moderate metabolic dysregulation during critical periods of visual development may be sufficient to disrupt emmetropization and ocular motor control.\u003c/p\u003e \u003cp\u003eOne of the most important contributions of this study is the derivation of phenylalanine cutoff values associated with multiple ocular abnormalities using ROC analysis. To our knowledge, no recent PKU studies have quantified specific phenylalanine thresholds for corneal, lenticular, retinal, and optic nerve changes within a single cohort. Our data support a multi-tiered model whereby ocular risk rises significantly above 500 \u0026micro;mol/L and becomes high above 1000 \u0026micro;mol/L\u0026mdash;information that has not been provided in previous ophthalmic or metabolic studies.\u003c/p\u003e \u003cp\u003eTogether, our findings reveal a consistent, multi-tissue dose\u0026ndash;response pattern: as phenylalanine levels rise, abnormalities appear progressively across corneal, pigmentary, lenticular, retinal, and optic nerve structures. Earlier studies examined each structure in isolation, but our integrated dataset demonstrates that the entire eye behaves as a metabolically sensitive organ. This unified pattern supports the development of ophthalmic screening strategies tailored to phenylalanine levels and may assist clinicians in identifying patients who require more frequent ocular monitoring.\u003c/p\u003e \u003cp\u003eIn summary, this study confirms that ocular involvement in PKU is metabolically driven, clinically meaningful, and detectable even in childhood. Elevated phenylalanine was consistently linked to structural abnormalities and functional visual loss. By integrating detailed ophthalmic evaluation with quantitative ROC-based phenylalanine thresholds, our study introduces novel evidence that may improve targeted ophthalmic surveillance and support more stringent metabolic management to prevent long-term visual morbidity in children with PKU.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOcular abnormalities are common and multisystemic in children with phenylketonuria and are associated with increasing serum phenylalanine levels. Exploratory analyses suggest that phenylalanine concentrations above approximately 500 \u0026micro;mol/L are associated with an increased likelihood of ocular involvement, with more severe abnormalities observed at levels exceeding 1000 \u0026micro;mol/L. These findings support the eye as a metabolically sensitive organ and highlight the potential value of phenylalanine-guided ophthalmic surveillance in pediatric PKU.\u003c/p\u003e"},{"header":"STUDY LIMITATION and FUTURE DIRECTIONS","content":"\u003cp\u003eThis study\u0026rsquo;s cross-sectional design and single-center cohort limit causal inference and generalizability. Visual assessments were sometimes challenging in younger children, and detailed imaging was not available for all participants. Longitudinal studies are needed to track the progression of ocular changes in PKU and evaluate the impact of early and sustained metabolic control. Advanced imaging and functional assessments could further clarify structure\u0026ndash;function relationships, while multicenter studies may validate phenylalanine thresholds for risk stratification and guide personalized ophthalmic screening protocols\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePKU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhenylketonuria\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePhe\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhenylalanine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReceiver Operating Characteristic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArea Under the Curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIOP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntraocular Pressure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003elogMAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLogarithm of the Minimum Angle of Resolution\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMS/MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTandem Mass Spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhenylalanine Hydroxylase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMHP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMild Hyperphenylalaninemia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecPKU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClassical Phenylketonuria\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emPKU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMild Phenylketonuria\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMUCH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMansoura University Children\u0026rsquo;s Hospital\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMOC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMansoura Ophthalmology Center\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e :This study was conducted by the principles outlined in the Declaration of Helsinki. The study protocol was approved by the Research Ethics Committee of Faculty of Medicine, Mansoura University (Code Number :R.23.12.2432) and followed by Institutional Research Board. Informed consents were obtained from all parents of the children included in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e : informed consent was obtained from all patient \u0026nbsp;caregivers before inclusion in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e:\u0026nbsp;The datasets generated and/or analyzed during the current study are not publicly available due to institutional and ethical restrictions but are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e:The authors declare that no conflicts of interest associated with this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch funding\u003c/strong\u003e: the authors declare that no funds ,grants, or other \u0026nbsp; \u0026nbsp;support were received during the preparation of this research .\u003cstrong\u003eAuthor contributions\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.E.\u003c/strong\u003e contributed to patient recruitment, clinical metabolic assessment, data collection, and drafted the initial manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.L.\u003c/strong\u003e performed comprehensive ophthalmological examinations and contributed to data acquisition and interpretation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM.A.H.\u003c/strong\u003e conceptualized and designed the study, performed statistical analysis, supervised the research process, contributed to data interpretation, and critically revised the manuscript for important intellectual content.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors approved the final manuscript and agreed to be accountable for all aspects of the work\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e: The authors would like to thank the children and their families for their participation in this study. We also acknowledge the nursing staff and laboratory personnel at Mansoura University Children\u0026rsquo;s Hospital and Mansoura ophthalmology center for their assistance in patient coordination and biochemical assessments.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWaisbren SE, Noel K, Fahrbach K, Cella C, Frame D, Dorenbaum A, et al. Phenylalanine blood levels and clinical outcomes in phenylketonuria: a systematic literature review and meta-analysis. Mol Genet Metab. 2007;92(1\u0026ndash;2):63\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomm A, Thiele AG, Rohde C, Kirmse S, Kiess W, Beblo S. Executive functions \u0026amp; metabolic control in phenylketonuria (PKU) and mild hyperphenylalaninemia (mHPA). Mol Genet Metab. 2024;143(1\u0026ndash;2):108544.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalter JH, White FJ, Hall SK, MacDonald A, Rylance G, Boneh A, et al. How practical are recommendations for dietary control in phenylketonuria? Lancet. 2002;360(9326):55\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoat SJ, Schulenburg-Brand D, Lemonde H, Bonham JR, Weykamp CW, Mei JV, et al. Performance of laboratory tests used to measure blood phenylalanine for the monitoring of patients with phenylketonuria. J Inherit Metab Dis. 2020;43(2):179\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamp KM, Parisi MA, Acosta PB, Berry GT, Bilder DA, Blau N et al. Phenylketonuria Scientific Review Conference: state of the science and future research needs. Mol Genet Metab. 2014;112(2):87\u0026ndash;122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Vliet D, van der Goot E, van Ginkel WG, van Faassen HJR, de Blaauw P, Kema IP, et al. The increasing importance of LNAA supplementation in phenylketonuria at higher plasma phenylalanine concentrations. Mol Genet Metab. 2022;135(1):27\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRondelli V, Koutsioubas A, Di Cola E, Fragneto G, Grillo I, Del Favero E, et al. Dysmyelination and glycolipid interference caused by phenylalanine in phenylketonuria. Int J Biol Macromol. 2022;221:784\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHimmelreich N, Shen N, Okun JG, Thiel C, Hoffmann GF, Blau N. Relationship between genotype, phenylalanine hydroxylase expression and in vitro activity and metabolic phenotype in phenylketonuria. Mol Genet Metab. 2018;125(1\u0026ndash;2):86\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHopf S, Nowak C, Hennermann JB, Schmidtmann I, Pfeiffer N, Pitz S. Saccadic reaction time and ocular findings in phenylketonuria. Orphanet J Rare Dis. 2020;15(1):124.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGramer G, Forl B, Springer C, Weimer P, Haege G, Mackensen F, et al. Visual functions in phenylketonuria-evaluating the dopamine and long-chain polyunsaturated fatty acids depletion hypotheses. Mol Genet Metab. 2013;108(1):1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo K, Zhou X, Chen X, Wu Y, Liu C, Kong Q. Expanded Newborn Screening for Inborn Errors of Metabolism and Genetic Characteristics in a Chinese Population. Front Genet. 2018;9:122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnwar MS, Waddell B, O'Riordan J. Neurological improvement following reinstitution of a low phenylalanine diet after 20 years in established phenylketonuria. BMJ Case Rep. 2013;2013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZwaan J. Eye findings in patients with phenylketonuria. Arch Ophthalmol. 1983;101(8):1236\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNulmans I, Lequeue S, Desmet L, Neuckermans J, De Kock J. Current state of the treatment landscape of phenylketonuria. Orphanet J Rare Dis. 2025;20(1):281.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerfozo C, Barta AG, Horvath E, Sumanszki C, Csakany B, Resch M, et al. Altered visual functions, macular ganglion cell and papillary retinal nerve fiber layer thickness in early-treated adult PKU patients. Mol Genet Metab Rep. 2020;25:100649.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerfozo C, Barta AG, Horvath E, Sumanszki C, Csakany B, Resch M, et al. Reduced macular thickness and macular vessel density in early-treated adult patients with PKU. Mol Genet Metab Rep. 2021;27:100767.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuonamassa R, Boscia G, Gaudiomonte M, Guerriero S, Fischetto R, Montepara A, et al. 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Invest Ophthalmol Vis Sci. 2016;57(10):4412\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Phenylketonuria, Ocular findings, Diet regiment","lastPublishedDoi":"10.21203/rs.3.rs-8929509/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8929509/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eAlthough phenylketonuria (PKU) is primarily recognized for its neurological sequelae, the extent and metabolic correlates of ocular involvement in early-treated pediatric patients remain insufficiently characterized. This study aimed to evaluate ocular findings in children with PKU and to examine their association with serum phenylalanine levels, with particular focus on quantitative risk stratification.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis cross-sectional study included 33 children less than 18 years diagnosed with phenylketonuria (PKU) by neonatal or selective screening and receiving regular follow-up at the Metabolic unit of Mansoura University Children\u0026rsquo;s Hospital, after obtaining informed parental consent. All participants underwent a comprehensive ophthalmological evaluation, including visual acuity assessment, cycloplegic refraction, anterior segment examination, fundus evaluation, intraocular pressure measurement, and ocular motility assessment. Serum phenylalanine levels were recorded, and receiver operating characteristic (ROC) curve analyses were performed to explore phenylalanine thresholds associated with ocular abnormalities.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOcular abnormalities were observed in a substantial proportion of participants and involved both anterior and posterior segments. Higher serum phenylalanine levels were significantly associated with increased likelihood of ocular involvement. ROC analysis identified phenylalanine ranges associated with elevated ocular risk, providing a quantitative framework for metabolic risk stratification..\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOcular abnormalities may occur in early-treated children with PKU and appear to be associated with phenylalanine control. Quantitative phenylalanine thresholds identified through ROC analysis may support risk-stratified ophthalmic surveillance strategies in pediatric PKU.\u003c/p\u003e","manuscriptTitle":"Phenylalanine-associated ocular risk stratification in early-treated children with phenylketonuria: a cross-sectional study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-26 16:52:39","doi":"10.21203/rs.3.rs-8929509/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"97a945ec-5209-4d2f-9149-8cfb52479e9b","owner":[],"postedDate":"March 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T07:43:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-26 16:52:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8929509","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8929509","identity":"rs-8929509","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}