Promise and challenges of dystonia brain banking: establishing a human tissue repository for studies of X-Linked Dystonia-Parkinsonism

Abstract

X-Linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease affecting individuals with ancestry to the island of Panay in the Philippines. In recent years there has been considerable progress at elucidating the genetic basis of XDP and candidate disease mechanisms in patient-derived cellular models, but the neural substrates that give rise to XDP in vivo are still poorly understood. Previous studies of limited XDP postmortem brain samples have reported a selective dropout of medium spiny neurons within the striatum, although neuroimaging of XDP patients has detected additional abnormalities in multiple brain regions beyond the basal ganglia. Given the need to fully define the CNS structures that are affected in this disease, we created a brain bank in Panay to serve as a tissue resource for detailed studies of XDP-related neuropathology. Here we describe this platform, from donor recruitment and consent to tissue collection, processing, and storage, that was assembled within a predominantly rural region of the Philippines with limited access to medical and laboratory facilities. Thirty-six brains from XDP individuals have been collected over an initial 4 years period. Tissue quality was assessed based on histologic staining of cortex, RNA integrity scores, detection of neuronal transcripts in situ by fluorescent hybridization chain reaction, and western blotting of neuronal and glial proteins. The results indicate that this pipeline preserves tissue integrity to an extent compatible with a range of morphologic, molecular, and biochemical analyses. Thus the algorithms that we developed for working in rural communities may serve as a guide for establishing similar brain banks for other rare diseases in indigenous populations.

Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat

“…Post-mortem brain tissue from XDP patients (n = 41; 40 with age at onset and death) was obtained in collaboration with the Collaborative Center for XDP (CCXDP), at MGH (Boston, MA, USA), Makati Medical Center (Makati City, Philippines), and the Sunshine Care Foundation (Panay, Philippines). Detailed descriptions of all methods related to donor consent, brain collection and tissue processing have been previously reported [ 44 ] and the use of XDP patient post-mortem brain tissue and all study procedures were approved by Institutional Review Boards at Makati Medical Center (Makati City, Philippines) and MGH (Boston, MA, USA). Genomic DNA was extracted from different brain regions using the DNeasy Blood and Tissue Kit (Qiagen), according to manufacturer’s instructions and with the following modifications: samples were incubated in buffer ATL and Proteinase K overnight at 56 °C; washes AW1 and AW2 were repeated; DNA was eluted in 100 μl of Qiagen Elution Buffer, preheated to 56°C, applied to the spin columns, and incubated at room temperature for 10 min before centrifugation.…”

Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat

“…Post-mortem brain tissue from XDP patients (n=41; 40 with age at onset and death) was obtained in collaboration with the Collaborative Center for XDP (CCXDP), at MGH (Boston, MA, USA), Makati Medical Center (Makati City, Philippines), and the Sunshine Care Foundation (Panay, Philippines). Detailed descriptions of all methods related to donor consent, brain collection and tissue processing have been previously reported [44] and the use of XDP patient post-mortem brain tissue and all study procedures were approved by Institutional Review Boards at Makati Medical Center (Makati City, Philippines) and MGH (Boston, MA, USA). Genomic DNA was extracted from different brain regions using the DNeasy Blood and Tissue Kit (Qiagen), according to manufacturer’s instructions and with the following modifications: samples were incubated in buffer ATL and Proteinase K overnight at 56°C; washes AW1 and AW2 were repeated; DNA was eluted in 100 μl of Qiagen Elution Buffer, preheated to 56°C, applied to the spin columns, and incubated at room temperature for 10 minutes before centrifugation.…”

Dissection of TAF1 neuronal splicing and implications for neurodegeneration in X-linked dystonia-parkinsonism

“…All procedures related to donor informed consent, tissue collection and distribution for research have been reported. 21 The other two brains were provided by Dr C. Klein (IDs L-10.322 and L-7.995). Specimens were used in accordance with the ‘Gesetz über das Leichen-, Bestattungs- und Friedhofswesen (Bestattungsgesetz) des Landes Schleswig-Holstein vom 04.02.2005, Abschnitt II, § 9 (Leichenöffnung, anatomisch)’.…”

“…Control tissues included PFC ( n = 3), MC ( n = 1), CB ( n = 2), AP ( n = 1), caudate nuclei (CN, n = 3) and thalamus (TH, n = 1). Hexamer repeat length for the processed XDP samples was genotyped by Fernandez-Cerado et al 21 and Reyes et al 22 and repeat numbers are as follows: 17.01, 44; 17.06, 41; 17.10, 34; 17.12, 42; 17.13, 46; 17.17, 54; L-7.995, 45; L-10.332, 41.…”