Between 1997 and 2023, a total of 146 individuals with a confirmed diagnosed of NPC1 were enrolled in the National Institutes of Health (NIH) IRB-approved NPC1 natural history studies (NCT00001367 and NCT00344331) after obtaining consent or assent from the individuals or their guardians. The purpose of initiating this study was to identify clinical or biochemical markers that could be used as outcome measures. Comprehensive visits were conducted at intervals of approximately 6-to-12 months assessing medical status, disease progression, and swallowing function. Visits included the following evaluations: history, physical exam, cranial nerve (oral-motor assessments) [27], and videofluoroscopic swallowing studies (VFSS), when clinically indicated.

Of the 146 enrolled individuals, 14 met inclusion criteria for our analysis, which required neurological onset after the age of 15 years and completion of swallowing evaluations. Patients who had received investigational treatments in development or through expanded access (Arimoclomol and Adrabetadex, respectively) were excluded from our analysis due to their unknown effects on swallowing. However, patients receiving miglustat were included due to its reported protective effect on swallowing function [4, 16, 20,21,22,23,24]. All individuals were assessed at baseline, and 6 (42.3%) were followed longitudinally for a total of 24 evaluations. Attempts were made for yearly follow-up in this study, but attrition was experienced with varying lengths of time between visits.

During each study visit, a standardized and NPC1-specific medical history was gathered by conducting interviews with the individual, parent or guardian and reviewing relevant medical records, if available. The baseline visit involved a comprehensive assessment covering various aspects such as phenotypic presentation, diagnostic history, seizure history, and pharmacological interventions. This information helped determine the onset of NPC1-specific symptoms, neurological symptoms, seizures, diagnostic delays, and miglustat use. For our analysis, we categorized peripheral disease symptoms as NPC1-specific symptoms [1, 5, 7,8,9, 14, 15, 19, 20] and symptoms originating from the central nervous system as neurological symptoms, excluding psychiatric symptoms.

Additionally, the NPC1 neurological severity score (NSS) was used to assess the severity of neurological symptoms. The NSS, based on Yanjanin et al.'s description [28], was scored based on information obtained from the interview conducted at each visit. It encompasses nine major domains (ambulation, cognition, eye movement, fine motor skills, hearing, memory, seizures, speech, and swallowing) and eight minor domains (auditory brainstem response, behavior, gelastic cataplexy, hyperreflexia, incontinence, narcolepsy, psychiatric issues, and respiratory problems). We specifically focused on domains that related to swallowing function, which included ambulation, speech, swallowing, fine motor skills, cognition, memory, seizures, cataplexy, narcolepsy, behavior, psychiatric issues, and respiratory problems. The total NSS was calculated by summing scores from all domains included in the scale.

Since dysarthria and dysphagia have been prominently noted in the AO cohort, a speech-language pathologist (SLP) at the NIH evaluated swallowing function at each participant’s visit. Additionally, individuals/proxies were interviewed by an SLP and completed questionnaires to identify possible triggers of dysphagia and determine if further assessment with a VFSS was necessary. Common triggers for VFSS included reported swallowing difficulties, intermittent coughing or choking, challenges swallowing various textures, and issues with bolus transit. VFSS was systematically conducted using various textures (liquid, puree, and solid), and swallowing function was evaluated with 26 parameters of impairments [11]. Two post-hoc interpretive scales, American Speech-Language-Hearing Association National Outcome Measures Scale (ASHA-NOMS) [29] and the NIH Penetration and Aspiration Scale (NIH-PAS) [30] were utilized to document a individual’s ability to swallow safely and identify aspiration risk. The ASHA-NOMS swallow domain, ASHA-NOMS dietary modification subdomain, and NIH-PAS were included in our analysis to examine AO NPC1 disease progression and note associations with other clinical measures. The scales for ASHA-NOMS, NIH-PAS, solid modifications, and liquid modifications were reversed to align with increasing severity direction and were then analyzed accordingly, similar to Solomon et al. [4].

In the NIH NPC1 natural history study, all but two AO patients underwent a lumbar puncture during the baseline visit to obtain a cerebral spinal fluid (CSF) sample (n = 12). In our analysis, we included the neurofilament light chain (NEFL) and ubiquitin carboxyl-terminal esterase L1 (UCHL1) levels in the CSF of a subgroup of patients (n = 9) who were previously measured in Agrawal et al. in 2023 and Cawley et al. in 2023 [31, 32].

Data are described using frequency (percentage) and descriptive statistics [mean ± SD or median (IQR)]. All data were assessed for distributional assumptions and analyzed accordingly. Categorical data between groups (e.g. sex, follow-up) were compared by Fisher’s Exact tests, and if ordinal (e.g., PAS, ASHA-NOMS) using the Cochran-Armitage Trend test. Continuous data were compared between groups by t-tests or Wilcoxon rank sum tests, as appropriate. Correlation analyses utilized Spearman’s rho. Longitudinal data were descriptive. Regression models assessed the relation between NEFL and UCHL1 and NPC1 outcomes with age as a covariate. Statistical evidence was assessed by magnitude of differences, data variability, and p-values. Data were analyzed using SAS v9.4 (SAS Institute, Inc, Cary, NC).