Magnetic resonance brain volumetry biomarkers of CLN2 Batten disease identified with miniswine model

Abstract

Late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease (Batten disease) is a rare pediatric disease, with symptom development leading to clinical diagnosis. Early diagnosis and effective tracking of disease progression are required for treatment. We hypothesize that brain volumetry is valuable in identifying CLN2 disease at an early stage and tracking disease progression in a genetically modified miniswine model. CLN2^R208X/R208X miniswine and wild type controls were evaluated at 12- and 17-months of age, correlating to early and late stages of disease progression. Magnetic resonance imaging (MRI) T1- and T2-weighted data were acquired. Total intercranial, gray matter, cerebrospinal fluid, white matter, caudate, putamen, and ventricle volumes were calculated and expressed as proportions of the intracranial volume. The brain regions were compared between timepoints and cohorts using Gardner-Altman plots, mean differences, and confidence intervals. At an early stage of disease, the total intracranial volume (- 9.06 cm³), gray matter (- 4.37% 95 CI - 7.41; - 1.83), caudate (- 0.16%, 95 CI - 0.24; - 0.08) and putamen (- 0.11% 95 CI - 0.23; - 0.02) were all notably smaller in CLN2^R208X/R208X miniswines versus WT, while cerebrospinal fluid was larger (+ 3.42%, 95 CI 2.54; 6.18). As the disease progressed to a later stage, the difference between the gray matter (- 8.27%, 95 CI - 10.1; - 5.56) and cerebrospinal fluid (+ 6.88%, 95 CI 4.31; 8.51) continued to become more pronounced, while others remained stable. MRI brain volumetry in this miniswine model of CLN2 disease is sensitive to early disease detection and longitudinal change monitoring, providing a valuable tool for pre-clinical treatment development and evaluation.

Figure 1

Exemplary T1-weighted (T1w) and T2-weighted (T2w) MRI data from a CLN2^R208X/R208X miniswine at 12-months-of-age (top left) and 17-months (top right), and a wild type (WT) comparator (bottom). Images were aligned using anterior commissure, posterior commissure, and basal pons, so that equivalent axial slices were taken from each time point.

Figure 2

The relationship between weight change (17–12 months) and change in intracranial volume (ICV) (17–12 months) in the CLN2^R208X/R208X miniswine (blue, R² = 0.0011) and WT miniswine (red, R² = 0.13), showing reduction in ICV (volume change range: − 1 to − 8 cm³) across CLN2^R208X/R208X miniswine unrelated to the large variation in weight change across the cohort (0 to 22 kg).

Figure 3

Early disease stage (at 12-months-of-age) ICV proportion comparison of brain regions between WT and CLN2^R208X/R208X cohorts. The average difference and 95% confidence interval (black circle with error bar in CLN2 minus WT column) do not cross the horizontal line at zero and indicate reliable measurement difference between the cohorts in gray matter (A), CSF (B), caudate (C), and putamen (D). When the average difference and 95% confidence intervals (black circle with error bar in CLN2 minus WT column) cross the horizontal line at zero as it does for white matter (E) and ventricles (F), an effect size equal to zero is possible, and reliable measured difference is unlikely.

Figure 4

Late disease stage (at 17-months of age) ICV proportion comparison of brain regions between WT and CLN2^R208X/R208X cohorts. The average difference and 95% confidence interval (black circle with error bar in CLN2 minus WT column) do not cross the horizontal line at zero and indicate reliable measurement difference between the cohorts in gray matter (A), CSF (B), caudate (C), and putamen (D). The average difference and 95% confidence intervals (black circle with error bar in CLN2 minus WT column) cross the horizontal line at zero for white matter (E) and ventricles (F), indicating reliable measured difference is unlikely.

Figure 5

ICV proportion comparison of brain regions between 12- and 17-month-old CLN2^R208X/R208X minipigs. The average difference and 95% confidence interval (black circle with error bar in 17 mos minus 12 mos column) do not cross the horizontal line at zero and indicates reliable measurement difference between the cohorts in gray matter (A), CSF (B), putamen (D), and ventricles (F). When the average difference and 95% confidence intervals (black circle with error bar in 17 mos minus 12 mos column) cross the horizontal line at zero as it does for caudate (C) and white matter (E), an effect size equal to zero is possible, and reliable measured difference is unlikely.

Figure 6

ICV proportion comparison of brain regions between 12- and 17-month-old WT miniswine. The average difference and 95% confidence interval (black circle with error bar in 17 mos minus 12 mos column) do not cross the horizontal line at zero and indicates reliable measurement difference between the cohorts in the putamen (D). When the average difference and 95% confidence intervals (black circle with error bar in 17 mos minus 12 mos column) cross the horizontal line at zero as it does for gray matter (A), CSF (B), caudate (C), white matter (E) and ventricles (F), an effect size equal to zero is possible, and reliable measured difference is unlikely.

Figure 7

Mitochondrial ATP synthase subunit c accumulates in the somatosensory cortex of CLN2^R208X/R208X animals. Subunit c accumulation shown in the somatosensory cortex at 17-months-of-age. Mean ± SEM, Nested t-test. ****p < 0.0001. Scale bar = 200 µm.