It should be noted
that information about family history was lacking in a significant proportion (23.7%) of the MCF FTLD-TDP cohort and these were included in the “sporadic” group. The MCF clinical ALS cohort represents a sequential series of 229 clinical ALS patients ascertained by the ALS Center at MCF. These patients underwent a full neurological evaluation including selleck inhibitor electromyography, clinical laboratory testing, and imaging as appropriate to establish the clinical diagnosis of ALS. A positive family history in the MCF ALS series was defined as a first- or second-degree relative with ALS. The Control cohort (n = 909) was comprised of DNA samples from 820 control individuals collected from the Department of Neurology and DNA extracted from 89 normal control brains from the MCF brain bank. The GGGGCC hexanucleotide Sunitinib repeat in C9ORF72 was PCR amplified in family VSM-20 and in all patient and control cohorts using the genotyping primers listed in Table S2 using one fluorescently labeled primer followed by fragment length analysis on an automated ABI3730 DNA-analyzer (Applied Biosystems). The PCR reaction was carried out in a mixture containing 1M betaine solution, 5% dimethylsulfoxide, and 7-deaza-2-deoxy GTP in substitution
for dGTP. Allele identification and scoring was performed using GeneMapper v4.0 software (Applied Biosystems). To determine the number of GGGGCC units and internal composition of the repeat, 48 individuals homozygous for different fragment lengths were sequenced using the PCR primers. To provide a qualitative assessment of the presence of an expanded (GGGGCC)n hexanucleotide repeat in C9ORF72, we performed
a repeat-primed PCR reaction in the presence of 1M betaine, 5% dimethyl sulfoxide and complete substitution of 7-deaza-2-deoxy GTP for dGTP using a previously optimized and described cycling program ( Hantash et al., 2010). Primer sequences not are provided in Table S2. PCR products were analyzed on an ABI3730 DNA Analyzer and visualized using GeneMapper software. A 241 bp digoxigenin (DIG)-labeled probe was generated using primers listed in Table S2 from 10 ng gDNA by PCR reaction using PCR DIG Probe Synthesis Kit Expand High fidelity mix enzyme and incorporating 0.35 mM DIG-11-dUTP: 0.65 mM dTTP (1:6) in the dNTP labeling mix as recommended in the DIG System User’s Guide (Roche Applied Science). A total of 2 μl of PCR labeled probe per ml of hybridization solution was used as recommended in the DIG System User’s Guide. A total of 5–10 μg of gDNA was digested with XbaI at 37°C overnight and electrophoresed in 0.8% agarose gels in 1× TBE. DNA was transferred to positively charged nylon membrane (Roche Applied Science) by capillary blotting and crosslinked by UV irradiation.