Phenylalanine hydroxylase (PAH) deficiency, conventionally identified as Phenylketonuria (PKU), is an autosomal recessive inherited metabolic disorder marked by various mutations in the gene (1). PAH serves as a catabolic enzyme responsible for converting L-phenylalanine (L-Phe) into L-tyrosine (L-Tyr), a process reliant on molecular oxygen, iron, and tetrahydrobiopterin (BH4) (2). Such deficiency leads to an impaired degradation of L-Phe and its metabolites, resulting in elevated levels in the blood. The hyperphenylalaninaemia and the increased Phe: Tyr ratio leads to Phe and Tyr to compete to cross the blood–brain barrier and tissues of affected individuals, resulting in an accumulation of Phe in the brain, especially in the White Matter (3). The prevalence of PKU varies worldwide, with an incidence of 1:10,000 newborn in Europe (4). As PKU has autosomal recessive inheritance, consanguineous marriage is an important risk factor; thus, high disease incidence is expectable for countries with a high rate of consanguineous marriages (5).

Untreated PKU can lead to a severe neurological impairment and progressive intellectual disability, accompanied by several additional symptoms such as eczematous rash, epilepsy, autism, seizures, and motor deficits (6). The current classification of PKU distinguishes less severe forms of PAH deficiency, referred to as moderate PKU, mild PKU, mild hyperphenylalaninaemia (HPA) or benign HPA, and more severe manifestations referred to as classic PKU (2). The neuropathology is determined by the increased levels of L-Phe, leading to disruptions in neuronal dendritic outgrowth and synaptic connectivity, as well as impaired cerebral glucose metabolism and neurotransmitters deficiency. The primary marker of disease progression includes white matter lesions (WMLs), which correlate with both metabolic control and the patient’s age (2).

To date, the mainstay of the treatment involves limiting dietary phenylalanine intake, coupled with daily supplementation of Phe-free amino acids. Indeed, the introduction of the neonatal screening allowed early intervention for this condition, with a consequent prevention of most of neuropsychological complications and an improved quality of life (3). By consequence, individuals diagnosed with PKU and subjected to lifelong therapy from birth have significantly reduced the incidence of the more severe impairments associated with untreated PKU. This specific group is termed early-treated PKU (ETPKU) (7).

In such a scenario, ocular findings include photophobia, ocular hypopigmentation, cataract and corneal opacities (8), and various functional visual impairment such as a lower Best-corrected visual acuity (BCVA) and a lower contrast sensitivity (9). The introduction of optical coherence tomography (OCT) has facilitated the identification of retinal neurodegeneration across various systemic and ocular conditions (10). Specifically, earlier researchers have directed their attention towards assessing the thickness of the retinal nerve fiber layer (pRNFL) and the ganglion cell layer (GCC), proposing these measurements as potential biomarkers for neurodegeneration (11).

Furthermore, due to the recent advancements in ophthalmic imaging techniques, notably OCTA, the ability to visualize vascular disorders has expanded to encompass various neurodegenerative conditions, including Parkinson’s disease (12) (PD).

Given the retina’s status as an extension of the central nervous system and shares numerous common characteristics with the central nervous system’s vasculature, the potential identification of retinal structural changes through OCT and vascular alterations using OCTA in ETPKU could potentially provide insights into overarching cerebral modifications that manifest during the initial stages of the disease. Consequently, the primary objective of this study was to explore changes in pRNFL thickness and the radial peripapillary capillary (RPC) plexus in individuals with ETPKU when contrasted with controls.

In this observational study, we exanimated the potential role of radial peripapillary capillary density and peripapillary retinal nerve fiber layer as markers of retinal damage in individuals with ETPKU compared to age-matched controls. Our findings align with previous research revealing a significant reduction in pRNFL thickness in ETPKU patients. Interestingly, we also observed that ETPKU patients exhibited a higher density of the total RPC compared to controls, suggesting an increased vascular activity associated with the disease.

ETPKU patients, thanks to neonatal screening and the early initiation of life-long phenylalanine-lowering therapy, benefit from protection against the most severe consequences of the disease (16). Nevertheless, ETPKU patients have more pronounced issues in social functioning and neurocognitive problems (17).

Recently, several researchers explored OCT and OCTA findings in ETPKU patients. For instance, Serfozo and coworkers observed significant thinning of both pRNFL and GCC in ETPKU compared to controls. This suggests that ocular microstructural abnormalities in PKU patients might be influenced by dietary control (13). Subsequently, Lotz-Havla and coworkers corroborated these findings. They also noted a reduction of the inner plexiform layer (IPL) volume, and an inner nuclear layer (INL) swelling strongly correlated with the metabolic control (18). These findings are partly consistent with our results, as we did not measure macular thickness, but we observed a thinning in the pRNFL thickness ETPKU patients who were solely treated with dietary control. Several studies have reported a significant correlation between pRNFL thinning and cerebral neurodegeneration, suggesting that average pRNFL might serve as a suitable biomarker for detecting and monitoring the progression of conditions such as Parkinson’s disease (PD) (19). The pattern of impairment observed in PKU, including ETPKU patients, shares similarities with other conditions characterized by dopamine imbalances and white matter damage such as PD or multiple sclerosis (MS) (20). In a recent review from Lancet, the authors stated that measurements of pRNFL and macular GCL and IPL combined could be an important tool for diagnosis, monitoring and research of MS (20). Specifically, in PKU and ETPKU patients, elevated phenylalanine (Phe) levels in the brain can inhibit the activities of enzymes like tyrosine and tryptophan hydroxylases activities, causing a reduction of dopamine (DA) and serotonin biosynthesis (21).

In the eye, DA is released by amacrine cells, activates dopamine receptors distributed throughout the retina, and covers multiple trophic roles related to circadian rhythmicity, cell survival, and eye growth (22). Consequently, conditions of metabolic dysregulation of DA like PKU, have been showed to be related to a DA imbalance in the retina as well (23). The exact mechanism of neurotoxicity on the retina in PKU is not clearly understood, but, according to one of the hypotheses, altered DA levels and impaired neurotransmission, are discussed to play a role in the retinal damage (21).

In addition to using OCT angiography, this study also evaluated the perfusion of the peripapillary vascular plexus. A previous OCTA study on ETPKU individuals by Serfozo et al. revealed a decrease in parafoveal superficial capillary plexus density in ETPKU compared to controls (24). However, our findings demonstrated a significant increase in RPC perfusion in ETPKU patients.

Given the aforementioned common pathways of retinal damage among DA imbalance diseases, it is not surprising the increased RPC density we found in ETPKU patients compared to controls (25). Indeed, the RPC is a network of capillaries that has a unique anatomic organization since it runs in parallel with retinal nerve fiber layer axon bundles and plays a role in nourishing the axons of retinal ganglion cells in peripapillary area (24).

We speculate that the neuroinflammation and the cellular stress may lead to an increased amount of vasodilatory and vasogenic mediators, resulting in enhanced blood flow to pRNFL axons. A similar mechanism has been proposed in Parkinson’s disease (26). This hypothetical condition might explain the simultaneous increase in RPC density and pRNFL thinning we found. Another potential explanation may be the formation of dysfunctional capillaries similar to the string vessels of Alzheimer’s disease.

The present study has limitations to consider when interpreting our findings. Our sample size was relatively small, which may limit the results generalizability. Furthermore, it’s important to acknowledge that our analysis focused solely on one phenotype of these patients (ETPKU), which may introduce a potential bias in our results. We analyzed OCTA images using spectral domain OCTA which utilizes shorter wavelength light in comparison with swept source OCT angiography. We did not measure the macular thickness and we did not compare the retinal measurements with brain parameters correlated to neurocognitive impairment. Additionally, we did not investigate the correlation between the duration of the pathology and the quantitative OCTA parameters analyzed.

Future randomized prospective case–control trials are necessary to confirm our findings, as well as longitudinal studies are always recommended for establishing the validity of such biomarkers.

In conclusion, our observational study provides evidence of neurovascular impairment in ETPKU patients who were solely treated with dietary control when compared to controls. Through structural OCT and OCT angiography, we observed thinning of the peripapillary nerve fibers accompanied by an increase in perfusion of the peripapillary vascular plexus. We suggest that, similar to other neurodegenerative diseases, increased neuroinflammation may lead to a higher production of vasodilatory mediators, resulting in an augmented vessel density in the RPC. These findings warrant further investigation as they may serve as an additional retinal biomarker in individuals with ETPKU.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants.

RB: Writing – original draft. GB: Formal analysis, Methodology, Supervision, Writing – review & editing. MGa: Data curation, Writing – original draft. SG: Supervision, Writing – original draft. RF: Data curation, Writing – original draft. AM: Conceptualization, Writing – original draft. MGr: Conceptualization, Writing – review & editing. MP: Data curation, Writing – original draft. PP: Conceptualization, Writing – original draft. EG: Conceptualization, Writing – review & editing. MD’a: Data curation Writing – original draft. GA: Conceptualization, Writing – review & editing. FB: Conceptualization, Writing – review & editing. PV: Supervision, Writing – review & editing.