Subjects were recruited from the National Neurological Institute “Carlo Besta”, Milan (Italy), the National Hospital for Neurology and Neurosurgery (London UK), the IRCCS Istituto Auxologico Italiano, Milan (Italy), and the Henri Mondor Hospital, Créteil (France). In total, samples from three Italian Cohorts (A,B for serum measurements; C for whole blood detection), one French Cohort (for detection in plasma) and three UK Cohorts (A, for plasma detection; B,C for whole blood) were collected and analyzed (see and ).
Clinical and genetic characteristics of HD, preHD and healthy patients.
All eligible participants received verbal and written information about the study, and provided signed, informed consent, according to the Declaration of Helsinki. Department of Neurodegenerative Disease, UCL Institute of Neurology University College London and National Hospital for Neurology and Neurosurgery, London: the study was approved by National Hospital of Neurology and Neurosurgery & Institute of Neurology Joint Research Ethics Committee (Central London REC 3). Centre National de référence maladie de Huntington, GHU Chenevier Mondor, Créteil: the study was approved by the CPP (Comité de protection des personnes) Henri Mondor, Créteil, France. National Neurological Institute-IRCCS “Carlo Besta”: the study was approved by the Istituto Neurologico “Carlo Besta” Institutional Review Board. IRCCS Istituto Auxologico Italiano, Milano: the study was approved by The Ethics Committees of IRCCS Istituto Auxologico Italiano “Comitato Etico dell'IRCCS Istituto Auxologico Italiano di Milano”.
Patients exclusion/inclusion criteria
Symptomatic HD patients
This group included patients that were positive on the molecular test for the presence of a CAG triplet with >35 repeats in the Huntington gene. They also manifested clinical signs and symptoms of HD. Clinical assessments of motor signs, disease staging and total functional capacity (TFC) were determined with the Unified Huntington's Disease Rating Scale (UHDRS). The age at onset was considered the time when clear motor signs and symptoms were first noticed.
We enrolled subjects that had more than 35 CAG triplet repeats in the Huntington gene, with no clinical symptoms or signs of the disease, and a UHDRS motor diagnostic confidence score of <4. For each pre-HD individual, the estimated probability of onset within the next 5 years was calculated on the basis of the number of CAG repeats and his/her present age, according to Langbehn 
This group included volunteers with no neurodegenerative disorders. Patients and controls were excluded from the study who were <18 years old and had known major medical conditions in addition to the primary genetic disorder.
Blood sampling and processing
For patients and control subjects from Henri Mondor Hospital, Créteil, and from the Neurological Institute-IRCCS “Carlo Besta” in Milan, blood samples were always withdrawn after an overnight fast. For patients from IRCCS Istituto Auxologico Italiano in Milan, blood samples were withdrawn after breakfast. From UCL Institute of Neurology, London, non-fasted samples were withdrawn.
French Cohort. 5 ml of blood were collected in EDTA-treated tubes between 9:00 and 12:00 a.m. Blood samples were maintained at room temperature (RT; 20–25°C) until plasma separation, which was performed up to 4 to 5 hours later. Tubes were centrifuged for 20 minutes at 3000×g in order to separate plasma from cellular components; plasma samples were stored in 1 ml aliquots at −80°C. Control, HD, and preHD subjects were recruited on the same day.
UK Cohort A. 5 ml of blood were collected in EDTA-treated tubes between 2:00 pm and 5:00 pm. Blood samples were maintained at RT until plasma separation, which was performed within 2 hours. Blood was transferred to a histopaque tube (A6929 ACCUSPIN™ System-HISTOPAQUE®-1077 Sigma Aldrich, St. Louis, MO, USA) and centrifuged for 10 minutes at 1000×g in order to separate plasma from cellular components; plasma samples were stored in 1 ml aliquots at −80°C and shipped in 200 µl aliquots.
Italian Cohort A. 3 ml of blood were collected between 9:00 and 12:00 a.m., after breakfast. Blood samples were maintained at RT for 1 hour, and serum was separated by centrifugation at RT for 10 minutes (2000×g). Serum samples were stored in 150 µl aliquots at −80°C.
Italian Cohort B. 3 ml of blood were collected between 9:00 and 12:00 a.m., after an overnight fast. Blood samples were maintained at RT for 1 hour, and serum was separated by centrifugation at RT for 10 minutes (2000×g). Serum samples were stored in 150 µl aliquots at −80°C.
UK Cohort B, C and Italian Cohort C. Whole venous blood was collected in an evacuated blood collection tube (PAXgene™ Blood RNA Tube, BD, Franklin Lakes, NJ, USA) that contained a stabilizing additive between 2:00 pm and 5:00 pm. The PAX tubes were left at room temperature for 2 hours as per Qiagen protocol and then stored at −80°C until RNA extraction.
Plasma and serum samples were assayed for BDNF protein levels with the BDNF Emax ImmunoAssay System (Promega, Madison WI, USA). In order to minimize inter-sample variance resulting from experimental variation, all samples were measured simultaneously and with BDNF ELISA kits from the same batch. Internal standards for the BDNF ELISA kit were used as suggested by 
and consisted of (a) a standard curve performed in each experiment to allow BDNF protein quantification, and (b) one standard sample present on all plates. Plasma and serum aliquots were thawed on the same day of the experiment at 9:00 a.m. Plasma was diluted at 1
20 in PBS1X; serum samples were diluted at 1
300 in PBS1X. Each plasma and serum sample was tested for BDNF content in three independent ELISA assays. Plasma and serum dilutions were tested in triplicate on each ELISA plate. A Synergy HT Multi-Detection Microplate Reader was used to read absorbance at 450 nm.
Total RNA was extracted from whole blood with the PAXgene™ Blood RNA System Kit (PreAnalytiX, Hombrechtikon, CH). Total RNA from brain was isolated from 200–300 mg of human brain tissue with 2 ml of TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). The tissue was then homogenised in liquid nitrogen with a mortar and pestle. To enhance the RNA yield, the samples were precipitated by adding 2 µl of glycogen solution (10 mg/ml) in isopropanol, and incubating at −80°C overnight. Total RNA concentration was measured with a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The RNA quality was verified by electrophoresis of 1 µg from each sample on an agarose gel. The total RNA was stored in aliquots at −80°C.
Reverse transcription of RNA
250 ng of total RNA was reverse-transcribed to single-stranded cDNA with Superscript III RNaseH- reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random primers in a volume of 20 µl, according to the manufacturer's instructions. Two independent reverse transcription reactions were performed for every RNA stock. For the analyses of BDNF mRNA isoforms in whole blood, 2 µg of total RNA was reverse-transcribed to single stranded cDNA. For the analyses of BDNF mRNA in the cortex, 1 µg of total RNA was reverse-transcribed to single stranded cDNA.
Three independent PCR analyses were performed for each of two independent reverse transcription reactions; thus, a total of six independent measurements were performed for each of the analysed mRNA. We used an iCycler Thermal Cycler with a Multicolour Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). All reactions were performed in a total volume of 25 µl that contained 5 µl of cDNA (1
10 dilution of reverse transcribed samples for BDNF, ROCK1, ANXA1, GAPDH, B2M and ß-actin amplification or 1
200 dilution of reverse transcribed samples for EAR amplification), 50 mM KCl, 20 mM Tris-HCI, pH 8.4, 0.2 mM dNTPs, 25 units/ml iTaq DNA polymerase, 3 mM MgCl2
, SYBR Green I with 10 nM fluorescein and stabilisers (iQTM SYBR Green Supermix-Biorad, Hercules, CA, USA), and 0.3 µM each of forward and reverse primers. The amplification cycles consisted of an initial denaturing cycle at 95°C for 3 minutes, followed by 45 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Fluorescence was quantified during the 60°C annealing step, and product formation was confirmed with a melting curve analysis (55°C–94°C).
The BDNF-specific primers were based on the BDNF coding sequence (GenBank accession number AF411339), as follows: BDNF 5′-TAACGGCGGCAGACAAAAAGA-3′; BDNF 5′-GAAGTATTGCTTCAGTTGGCCT-3′. The amplification product was 101 base pairs (bp) long. The expressed Alu repeat (EAR)-specific primers were based on the human interspersed Alu repetitive sequence. The primer sequences were: EAR 5′-GAGGCTGAGGCAGGAGAATCG-3′ EAR 5′-GTCGCCCAGGCTGGAGTG-3′. The amplification product was 87 bp long. The ANXA1 specific primers were set up referring to GenBank accession number NM_000700.1. The primers sequences were Fw: 5′- GAGCCCCTATCCTACCTTCAAT-3′; Rev: 5′-GATGGTTGCTTCATCCACACCT-3′. The amplification product was 88 bp long. The ROCK1 specific primers were set up referring to GenBank accession number NM_005406.2.
The primers sequences were Fw: 5′-AGGTTAGGGCGAAATGGTGTAGAA-3′; Rev: 5′-CCTCTCCTTTATCTTCTTCCAAGTC-3′. The GAPDH specific primers were set up referring to GenBank accession number NC_000012.10. The primer sequences were: GAPDH Fw: 5′-AGCTGAACGGGAAGCTCACT-3′ GAPDH Rev: 5′-AGGTCCACCACTGACACGTTG-3′. The amplification product was 67 bp long. The B2M specific primers were set up referring to GenBank accession number NC_000015.8. The primer sequences were: B2M Fw: 5′-GGAGAGAGAATTGAAAAAGTGGAGC-3′; B2M Rev: 5′-GGCTGTGACAAAGTCACATGGTT-3′. The amplification product was 143 bp long. The ß-actin specific primers were set up referring to GenBank accession number NC_000007.12. The primer sequences were: Fw: 5′- AGTGTGACGTGGACATCCGCA-3′; Rev: 5′- GCCAGGGCAGTGATCTCCTTCT-3′. The amplification product was 112 bp long.
Semi-quantitative PCRs were performed in a total volume of 50 µl, consisting of 100 ng of cortex cDNA or 200 ng of blood cDNA, 20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 µM each of forward and reverse primers, and 2.5 units of Taq polymerase (Invitrogen). The amplification cycles consisted of an initial denaturing cycle at 94°C for 5 minutes, followed by 35 cycles (or 40) of 1 minute at 94°C, 45 s at different temperatures, according to the melting temperature of the primers, and 45 s at 72°C. The sequences of all primers, their annealing temperatures, and the lengths of the PCR products are provided upon request.
As BDNF values do not have a normal distribution in both control and HD populations, the non-parametric Kruskal-Wallis test followed by Dunn's Multiple comparison test or Mann-Whitney two- tailed U-test were used for the statistical analyses, with significance being set at P<0.05. Median values were chosen because of the skewed distribution of the data. In the box plots, the boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. When 10 or more samples were analyzed, whiskers appear above and below the box to indicate the 90th and 10th percentiles. Unpaired t-test and Spearman's rank correlation coefficient were also used.