Supplementary information: Family history
Parents of generation I: Father died at early age, sudden death, was an otherwise healthy farmer. Mother died at age of 80, owned a shop after death of her husband, was seemingly healthy.
I.1: 87 years old, diagnosed with Alzheimer’s dementia at the age of 85.
I.2: Memory problems and possibly speech disorder, age of onset in early forties. Died at age of 50.
I.3: Reportedly healthy, died at advanced age.
I.4: Committed suicide at the age of 52. Took over the shop from her mother, but difficulty managing, developed memory problems and became slower, age of onset unknown.
II.1: Committed suicide around 60 years of age.
II.2: See main text, mother of the proband. Was diagnosed with dementia and epilepsy at the age of 47, died when 56 years old.
II.3: 64 years old, healthy.
II.4: See main text. 65 years old. Depression, memory problems with attention deficit and loss of impulse control. MRI brain show biparietal atrophy. Muscle biopsy might show some atypical lipofuscin-type inclusions on EM. Age of onset in mid-fifties.
II.5: 56 years old, reportedly healthy.
II.6: 50 years old, diagnosed with bipolar disorder at the age of 40. Stuttering speech.
II.7: 49 years old, healthy.
II.8: 42 years old, has the impression he has become more forgetful, started several years ago.
Otherwise healthy.
III.1: Healthy.
III.2: Index case, see main text.
Supplementary Materials and Methods
Patients and family members.
The index patient and family members were referred to the Movement Disorders Clinic in the Neurology Department at the University Hospital of Ghent in Belgium for clinical evaluation and diagnostic investigations. Genetic diagnostic testing and clinical genetic counselling were coordinated at the Center for Medical Genetics at the University Hospital of Ghent in
Belgium.
Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed paraffin-embedded 4 µm sections using antibodies anti-p62 (1:4000, guinea pig polyclonal; Progen Biotechnik GmBh, Heidelberg, Germany) and anti-phospho-TDP-43 (1:2000, mouse monoclonal, pS409/410;
Cosmo Bio Co. Ltd, Tokyo, Japan). For visualization species-appropriate DAKO EnVision TM kit with peroxidase-DAB system was used. Autofluorescence of the stored material was detected via filter block with an excitation wavelength of 400–440 nm (fluorescence
microscope Nikon E800, filter block BV-2A).
Electron microscopy
Biopsy samples of brain cortex, skeletal muscle and skin were processed according to standard methods for electron microscopy. After fixation in 4% glutaraldehyde and postfixation in 2% osmium tetroxide, blocks were embedded in araldite. Ultrathin epoxy sections were stained with uranyl acetate and lead citrate and examined by a FEI CM10 Philips electron microscope.
Exome sequencing
We performed DNA enrichment by SureSelect Human All Exon 50 Mb Kit, using 2 ug of DNA from 2 affected (II.4, III.2) and one unaffected (II.3) family member according to the manufacturer’s protocol. DNA sequencing was performed on the captured DNA library with
SOLiD 4 (Applied Biosystems, Carlsbad, USA) at Institute for Inherited Metabolic Disorders in Prague. We aligned reads in color space to the reference genome (hg19) by using
NovoalignCS version 1.01.08 (Novocraft, Malaysia). Sequence variants in the analyzed sample were identified with the SAMtools package (version 0.1.8)1. The high-confidence variants list (SNP quality > 100 and indel quality > 50) was annotated using GEMINI software v. 0.20.02.
C9orf72 analysis and short-repeat PCR
C9orf72 repeat expansion testing was performed by repeat-primed PCR, as described 3. We assayed the repeat expansion with a repeat-primed PCR as described elsewhere 4,5 with a gene-specific fluorescently labelled forward primer in C9orf72 exon 1a, a first reverse primer consisting of four GGGGCC repeats units, and an anchor sequence and a second reverse primer composed of the anchor sequence. Testing for the presence of a ‘short’ repeat (< 80 U) was performed by the ‘short-repeat PCR’ as described 6. The KAPA2G Robust HotStart DNA polymerase (Kapa Biosystems) was used to amplify repeat expansions in the short pathogenic size range. 50 ng genomic DNA was amplified using a fluorescently labelled primer pair flanking the repeat (5’-TCCTCACTCACCCACTCG-3’ and 5’-
ACTCGCTGAGGGTGAACAAG-3’), buffer A, 2.5 M betaine, 0.5µM of primers, 1.88 mM MgCl2 and 0.65mM dNTP. The PCR cycling profile was as follows: denaturation at 95°C for 5 minutes, 35 cycles at 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 1.5 minute;
and a final extension at 72°C for 3 minutes. The resulting PCR products were size-separated on agarose gel, analyzed on an ABI 3730 automated sequencer (Applied Biosystems) with GENESCAN LIZ600 as a size standard (Applied Biosystems) and genotypes were assigned using the in-house developed TracI genotyping software
(http://www.vibgeneticservicefacility.be).
References
1. Li H, Handsaker B, Wysoker A, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–2079.
2. Paila U, Chapman BA, Kirchner R, Quinlan AR. GEMINI: Integrative Exploration of Genetic Variation and Genome Annotations. Gardner PP, editor. PLoS Comput Biol.
2013;9:e1003153.
3. Gijselinck I, Van Langenhove T, van der Zee J, et al. A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study.
Lancet Neurol. 2012;11:54–65.
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5. Warner JP, Barron LH, Goudie D, et al. A general method for the detection of large CAG repeat expansions by fluorescent PCR. J Med Genet. 1996;33:1022–1026.
6. Gijselinck I, Van Mossevelde S, van der Zee J, et al. The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter. Mol Psychiatry. 2015;21:1–13.
7. Molinuevo JL, Blennow K, Dubois B, et al. The clinical use of cerebrospinal fluid biomarker testing for Alzheimer’s disease diagnosis: a consensus paper from the Alzheimer’s Biomarkers Standardization Initiative. Alzheimers Dement. 2014;10:808–
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