Retinal axonal degeneration in Niemann–Pick type C disease

Twelve NPC1-P with biallelic NPC1 gene mutations and two patients with one identifiable mutation, a classical or variant filipin test in cultured fibroblasts and a typical NPC phenotype (vertical supranuclear saccade palsy, cerebellar ataxia, dysarthria, cognitive impairment, dystonia; mean age 24 [range 9–38]; 8 male) were recruited from the university hospital specialized unit (Table 1). Mean duration of the disease was 14 ± 6.5 (SD) years. Seventeen of their asymptomatic first-degree family members, all confirmed heterozygous NPC1-MC (i.e., carrying one muted allele; mean age 50 [range 21–65]; 8 males) were included (Table 2). No neurological comorbidity was diagnosed based on a detailed examination by experts in movement disorders (TBE and SAS). The motor examinations were not evaluated blinded. Two HC groups age-matched for patients and carriers were also enrolled (14 HC for NPC1-P: mean age 27 [range 12–38] 7 males; 17 HC for NPC1-MC: mean age 51 [range 21–66]; 9 male). In the NPC1-P group, only 26 of the 28 eyes could be examined. Two patients discontinued the examination incompletely after examination of the first eye. Eight out of fourteen patients suffered from an organic psychiatric disorder due to NPC disease and took antipsychotic drugs. To our knowledge, there is no evidence of an effect of psychiatric medication on the OCT data shown here. This also applies to the anticonvulsive drugs. Subjects, and single eyes, respectively, with concomitant potentially OCT-confounding diseases (glaucoma, diabetes mellitus, retinal surgery, retinal disease, and ametropia > 6 diopters) were also excluded. Evaluation criteria for this study were retinal thickness/volume and visual disability measured by high and low contrast visual acuity (VA). Patients and mutation carriers underwent neurological examination and demographic information was obtained as summarized in Tables 1 and 2. Other causes that could explain motor anomalies were excluded by anamnesis and examinations as needed. Written informed consent was obtained from all patients participating in the study. The study was performed in accordance with the Helsinki II Declaration and approved by the ethics committee of the Ludwig-Maximilians-University, Munich, Medical Faculty (part of project no 280-16). All participants (or their legal representatives) gave written informed consent.

OCT examination was performed using an SD-OCT (Spectralis, Heidelberg Engineering, Heidelberg, Germany) with automatic real-time (ART) function for image averaging. We acquired peripapillary retinal nerve fiber layer thickness (pRNFL), macular RNFL (mRNFL), and volumes of combined ganglion cell and inner plexiform layer (GCIP), and combined outer plexiform layer and outer nuclear layer (OPONL). All macular layers were calculated as a 3 mm-diameter cylinder around the fovea from a macular volume scan (20° × 20°, 25 vertical B-scans, ART ≤ 49). The peripapillary RNFL (pRNFL) was measured with activated eye tracker using 3.4 mm ring scans around the optic nerve (12°, 1536 A-scans, ART ≤ 100). Segmentation of all layers was performed semi-automatically using software provided by the OCT manufacturer (Eye Explorer 1.9.10.0 with viewing module 6.3.4.0, Heidelberg Engineering, Heidelberg, Germany). One experienced rater (JH), blinded for the diagnosis, carefully checked all scans for sufficient quality and segmentation errors and corrected if necessary. OCT data in this study are reported and analyzed according to the APOSTEL and OSCAR-IB recommendations [18, 19]. The data were correlated with VOG measures and clinical scores.

Since patients with NPC demonstrate characteristic ocular motor deficits, including, but not limited to vertical supranuclear saccade palsy (VSSP), video-oculography (VOG) was performed in both NPC1-P and NPC1-MC, using a video-based eye-tracker system (EyeSeeCam^®, Munich, Germany) as previously described [20, 21]. Briefly, reflexive saccades that reflect brainstem function and smooth pursuit for cerebellar function were assessed.

To assess the overall neurologic status in NPC disease, the modified Disability Rating Scale (mDRS) by Pineda et al. [22] was applied; the mDRS is a four-domain scale (ambulation, manipulation, language, and swallowing) in an extended form [23], which also includes seizures and ocular movements, that assesses the severity of the disease and monitors the effect of treatment. All patients were examined by the same experienced neurologist (TBE) to reduce inter-rater variability. Cerebellar function was evaluated by administrating the Scale for the Rating and Assessment of Ataxia (SARA) [24] an eight-item clinical rating scale (gait, stance, sitting, speech, fine motor function, and taxis; range 0–40, with 0 being the best neurological status and 40 the worst). Moreover, NPC1-P were assessed by the Spinocerebellar Ataxia Functional Index (SCAFI), comprising the 8-m-Walking-Test (8MW), performed by having patients walking twice as quickly as possible from one line to another excluding turning, 9-Hole-Peg-Test (9HPT), and the number of “PATA” repetitions over 10 s (PATA) [25]. NPC1-MC were clinically assessed by an experienced neurologist in the field of movement disorders (SAS) with a particular focus on signs and symptoms of NPC disease: 1 point each was given for presence of a reduced arm swing, intention tremor, increased muscle tone, ankle clonus, and/or gait abnormalities resulting in a final motor score [26]. Scores ≥ 1 were considered pathological.

For statistical analysis, we evaluated 26 eyes of 14 NPC1-P and 34 eyes of 17 NPC1-MC. These eyes were compared with 28 eyes of 14 age- and sex-matched HC. NPC1-P had a significantly lower mRNFL (mean ± SD, 0.13 ± 0.01 mm³) and GCIPL (0.60 ± 0.05 mm³) compared to HC (mRNFL 0.14 ± 0.02 mm³; p = 0.01; GCIP 0.62 ± 0.04 mm³; p = 0.04) (Fig. 1). pRNFL showed a trend, but the difference was non-significant (98.12 ± 11.46 µm vs. 104.61 ± 8.39 µm; p = 0.06). No significant differences were observed for PMB, TMV, and OPONL. Furthermore, there were no significant differences between NPC1-MC and HC for all studied parameters (summarized in Table 3). An orienting comparison of visual acuity data showed a reduced Snellen Visual Acuity Equivalent (SVAE) and a reduced number of recognized letters (100%/2.5% Low-Contrast Sloan Letter Chart Testing (2 m), see Table 3) in the group of NPC1-P. A statistical analysis was not performed for unequally distributed missing VA values.

In NPC1-P, mean mDRS score correlated only with mean OPONL (ρ = − 0.617, p < 0.05). Mean SARA score was negatively related to GCIP (ρ = − 0.622, p < 0.05) (Fig. 2a, b). In NPC1-MC, motor scores (Table 1) were negatively associated with mean pRNFL (Average: ρ = − 0.719; p < 0.01) (Fig. 2c). No further correlations with clinical scores were seen.

Amplitude of upward vertical saccades to 20° stimulus in NPC1-P showed positive associations with averaged pRNFL (ρ = 0.645, p < 0.05) and averaged GCIP (ρ = 0.609, p < 0.05) (Table 1, Fig. 3a, b). Mean peak velocity of horizontal saccades to 30° stimulus correlated with averaged pRNLF (ρ = 0.650, p < 0.05) and averaged GCIP (ρ = 0.750, p < 0.01) (Table 1, Fig. 3c, d). Mean duration of horizontal saccades correlated negatively with pRNLF (ρ = − 0.727, p < 0.01) (Table 1, Fig. 3e). Averaged GCIP tracked with vertical smooth pursuit gain (ρ = 0.622, p < 0.05) (Table 1, Fig. 3f). In NPC1-MC, there were no significant associations between the retinal and video-ocular motor measures (data not shown).