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Figure S7. Nucleotide excision repair is not affected in C9ORF72 human fibroblasts. A) Dose-response curve for 4 healthy control human fibroblast lines (81E253, 86E1375, 06E0717 and 99E0774) and 4 C9ORF72 human fibroblast lines (13E634, 13E659, 17E0225, 17E0278) and the NER deficient XP25RO human fibroblast line treated with increasing dose of UV-C light (0–12 J/m2). B) Immunofluorescence staining showing the recruitment of NER factors XPC, XPB, XPA, XPF and XPG to local DNA damage (visualized by CPD antibody), induced by 60 J/m2 UV-C irradiation through a microporous filter. Fibroblast lines used for pictures: 13E634, 13E659 and 81E253 C) Human fibroblasts lines were treated with 16 J/m2 UV-C light and incubated with EdU for 1 h to measure unscheduled DNA synthesis (UDS) as measure of DNA repair. The NER-deficient XPC25RO cell line is shown as negative control. Ctrl 1–4 are lines 81E253, 86E1375, 06E0717 and 99E0774 in this order. C9 1–4 are lines 13E634, 13E659, 17E0225, 17E0278 in this order. (PDF 934 kb)

Figure S6 Validation of HR23B pathology by a second independent antibody. HR23B staining using Abcam antibody in C9ORF72 FTD cases (n = 5) and non-demented control (n = 3) post-mortem brain sections. Staining pattern is consistent with HR23B GeneTex antibody (see Figs. 1 and 2). All scale bars are 20 μm (PDF 1543 kb)

Figure S4 Pur-alpha staining in C9FTD cases and non-demented controls. Pur-alpha staining reveals abundant stress granules in both C9ORF72 FTD cases (n = 5) and non-demented controls (n = 3) post-mortem brain sections. All scale bars are 20 μm. (PDF 1826 kb)

Table S1. Neuropathological scores of C9ORF72 FTD patients. Neuronal loss score was based on hematoxylin and eosin (HE) staining and pathological report and scored as absent (0), mild (1), moderate (2) or severe (3). Pathological scores were based on the degree of pathology as absent (0), rare (1), occasional (2), moderate (3), or numerous (4). Brain areas: F = frontal cortex, T = temporal cortex, M = motor cortex, H = hippocampus dentate gyrus, C = cerebellum. NCI = neuronal cytoplasmic inclusion, NII = neuronal intranuclear inclusion, DNs = dystrophic neurites. (DOCX 23 kb)

Figure S3. FMRP staining in C9FTD cases and non-demented controls. FMRP staining does not reveal any differences between C9ORF72 FTD cases (n = 5) and non-demented control (n = 3) post-mortem brain sections. Occasional inclusions are found in the hippocampus dentate gyrus in both C9FTD cases and controls. All scale bars are 20 μm. (PDF 1883 kb)

Figure S2. ADARB2 staining in C9FTD cases and non-demented controls. A) Staining of ADARB2 in C9ORF72 FTD cases (n = 5) and non-demented control (n = 3) post-mortem brain sections shows some intranuclear inclusions in hippocampus CA and DG. All scale bars are 20 μm B) Immunofluorescence staining of ADARB2 (red) and p62 (green) in hippocampal dentate gyrus reveals ADARB2 punctuated staining and some intranuclear inclusions in both C9ORF72 FTD cases (n = 5) and non-demented controls (n = 3). Scale bars in fluorescent pictures are 10 μm. (PDF 2721 kb)

Figure S5. HR23B pathology in different brain areas of C9FTD cases. Staining of HR23B in several brain areas of C9FTD cases and non-demented controls. Pathology burden was highest in cortices (frontal, temporal and motor) and was mostly cytoplasmic (inclusions and neuropils) and intranuclear (cateye). Hippocampus dentate gyrus (DG) harbors perinuclear inclusions, and hippocampus cornu ammonis (CA) had some cells with strong nuclear staining. Pathology was low in cerebellum granular layer with only some nuclear and perinuclear inclusions and almost absent in cerebellum molecular layer. Our C9FTD cases did not show HR23B pathology in spinal cord neurons. All scale bars are 20â Îźm (PDF 2260 kb)

Figure S1. Ran-GAP staining in C9FTD cases and non-demented controls. Ran-GAP is predominantly localized to the nucleus and nuclear membrane. Unevenly shaped nuclear membranes occur in both C9ORF72 FTD cases (n = 5) and non-demented controls (n = 3). All scale bars are 20 μm. (PDF 2202 kb)

Table S2. Co-localization of HR23B with DPRs/pTDP-43/p62 differs between brain areas. The percentage is the amount of HR23B inclusions also positive for other pathological hallmarks (not the other way around). For example 11/153 for poly-GA in C9FTD patient 1 means that out of 153 HR23B inclusions, 11 were also positive forpoly- GA, which is 7%. F = frontal cortex. H = hippocampus dentate gyrus. *1 = All co-localizations are fibrils of poly-GP and HR23B in frontal cortex. Perinuclear inclusions of poly-GP did not stain positive for HR23B. *2 = In total 2 poly-PA inclusions have been found in frontal cortex of 5 C9FTD patients, too less to quantify. We therefore state N/A = non-applicable in this table. *3 = Non-demented cases had some p62 and some HR23B inclusions per person per section, which sometimes overlapped. No pTDP-43 inclusions were found so no co-localization of pTDP-43 with HR23B in non-demented cases. (DOCX 19 kb)

background: Fragile X syndrome (fxs) is the most common hereditary cause of intellectual disability and autism spectrum disorders.Targeted treatment is currently lacking. In the past decades an enormous amount of knowledge has been obtained concerning the involved molecular pathways, introducing potential targets for disease modifying therapy. aim: To present an overview of the development of targeted treatment for fxs. method: Several important publications were collected and indexed. results While preclinical animal model studies with targeted interventions are promising,thetranslationtothe clinic has been disappointing. conclusion: Targeted treatment for fxs is necessary and could be applied in other causes of autism spectrum disorders and intellectual disability. Factors relating to translation, study design and outcome measures are possibly contributing to the disappointing results. The clustering of patient care in a center of expertise is required to clinically implement future therapeutic strategies and to facilitate research. In addition, this improves patient care, one example being the recent medical guideline for children with fxs.

Table S1. Coding variants under the linkage peaks for LVH proxy measurements. Table S2. Selected damaging variants in the coding regions contained in the linkage regions. Table S3. SKAT and burden tests for genes of interest. Table S4. Results of linkage analyses before (LOD1) and after (LOD2) regression on GWAS SNPs under the linkage peaks. Table S5. Descriptive statistics of the Rotterdam study population. Table S6. Replications results in the Rotterdam Study. Figure S1. Venn diagram showing the overlap between the different ERF genotyping experiments. Figure S2. Pedigrees segregating rs138968470. (DOCX 119 kb)