B20-08 Pupillary Light Reflex Dynamics by PHOX2B Genotype in Congenital Central Hypoventilation Syndrome: Validation, Reliability, and Longitudinal Mixed-effects Analyses

Rationale Congenital central hypoventilation syndrome (CCHS) arises from PHOX2B gene mutations, leading to pervasive but variable autonomic dysregulation. Quantitative pupillometry offers a window into this physiology by measuring pupillary light reflex (PLR) dynamics, which are blunted in amplitude and velocity in children with CCHS compared with healthy controls. In contrast, PLR in healthy controls evolves with age. We hypothesized that PLR velocity and amplitude metrics capture the gradient of PHOX2B-related disease severity and remain stable across age, providing a developmentally invariant biomarker. We focused on the most common PHOX2B polyalanine repeat mutation (PARM) genotypes (20/25, 20/26, 20/27) and grouped analyses by variant severity (expected protein dysfunction) across a range of PHOX2B variants (including non-PARMs; NPARMs) to distinguish physiologic patterns. Methods We analyzed 544 bilateral PLR trials (1,088 eyes) from 54 PHOX2B-genotyped individuals with CCHS between 2010-2025. Pupillometry (handheld NeurOptics PLR-2000; Irvine, CA) measured PLR after a fixed white light stimulus (180 μW for 31 ms) after dark adaptation. Eight PLR metrics were extracted: initial diameter (INIT), minimum diameter (END), percent constriction (DELTA), latency (LAT), average/maximum constriction velocities (ACV/MCV), average dilation velocity (ADV), and time to 75% re-dilation (T75). Linear mixed-effects models included fixed effects for Genotype, Age, and Age×Genotype with subject random intercepts; first and second eyes were modeled separately for order effects. Variants were examined individually and as Moderate (20/25, 20/26, NPARM-Moderate) vs. Severe (20/27, 20/33, NPARM-Severe) groups. Left-right symmetry, order effects, Passing-Bablok, Bland-Altman, and within-week test-retest ICCs were assessed. Results Genotype influenced multiple PLR parameters, with largest effects observed for constriction velocities (ACV F = 28.5, MCV F = 25.4, both p < 0.0001) and percent constriction (DELTA F = 18.2). Moderate genotypes (20/25, 20/26, NPARM-Moderate) showed larger baseline pupil diameter (INIT) and faster, greater constriction than severe genotypes/variants (20/27, 20/33, NPARM-Severe), consistent with milder parasympathetic dysfunction. Group contrasts remained highly significant for ACV, MCV, and DELTA (all p < 0.001), while smaller differences were noted for END (p = 0.0375) and INIT (p < 0.001). Age effects and Age×Genotype interactions were NS. Measurement validation confirmed excellent symmetry (INIT r = 0.93; ACV r = 0.92), and high within-week reliability for INIT, ACV, and MCV (ICC≈0.91-0.96). Conclusions Pupillometry captures the physiologic gradient of autonomic dysfunction in CCHS, providing a window into PHOX2B-determined severity. Velocity and constriction magnitude (ACV, MCV, DELTA) emerge as indicators of parasympathetic impairment that complement PHOX2B variant stratification. Because these metrics are objective, rapid, and age-independent, pupillometry-derived PLR could offer remote disease monitoring, aid therapeutic trials, and support emerging gene or neurostimulation-based interventions.