br Materials and Methods br Molecular Diagnosis

Materials and Methods
Molecular Diagnosis of Prader–Willi Syndrome Genomic DNA was treated with sulfite using the CpGenome Turbo Bisulfite Modification Kit (Millipore, CA, USA) according to the manufacturer\'s instructions. Using methylation-specific primers in the CpG island of the SNRPN gene (Kubota et al., 1997), different amplicons were obtained from paternal and maternal chromosomal DNA in the core region associated with PWS. Two amplicons of different sizes were amplified from paternal and maternal chromosome in control samples. No amplification occurred in untreated samples. Only the amplicon from the mother was detectable in the PWS patient samples. The methylation-specific and non-methylation primers are as follows (Askree et al., 2011): MF 5′-TAAATAAGTACGTTTGCGCGGTC-3′ and MR 5′-AACCTTACCCGCTCCATCGCG-3′ were used to generate the 174bp methylation product. Primers, PF 5′-GTAGGTTGGTGTGTATGTTTAGGT-3′ and PR 5′-ACATCAAACATCTCCAACAACCA-3′ were used to amplify 100bp of the non-methylated allele. The PCR reaction mixture was prepared as follows: Taq-HS (TaKaRa, Tokyo, Japan) 12.5μL, primer forward 0.8nmol, primer reverse 0.8nmol, template DNA 60ng, ddH2O to a total volume of 25μL. The PCR program was 95°C for 5min, 95°C for 30s, 61°C for 30s, 72°C for 30s, cycled 40 times, 72°C for 7min. Electrophoresis was performed using a 3% agarose gel (Fig. S1).
Metabolomic Analysis The fecal metabolite extraction (Wu et al., 2010), and the d-tubocurarine sample preparation (Jiang et al., 2012; Xiao et al., 2009) were performed as described previously. All one-dimensional (1D) 1H NMR spectra of fecal water and urine samples were acquired on a Bruker AVIII 600MHz NMR spectrometer equipped with a cryogenic probe (Bruker Biospin, Germany). The first increment of NOESY pulse sequence was employed with continuous-wave irradiation on water peak during recycle delay and mixing time for water suppression. The 90° pulse was adjusted to about 10μs, and 64 scans were collected into 32k data points for each spectrum with the spectral width of 20ppm. To assist metabolite assignments, two-dimensional (2D) NMR spectra were acquired including 1H–1H COSY, 1H–1H TOCSY, 1H J-resolved, 1H–13C HSQC and 1H–13C HMBC for typical samples. Fourier transformation of the free induction decays was performed after multiplying by an exponential function with a line-width factor of 1Hz. The phase- and baseline-corrections were achieved manually, and chemical shift was calibrated to the TSP signal at δ0.00 with software TOPSPIN (v3.0, Bruker Biospin). For the spectra of fecal water, the spectral region δ0.5–9.5 was integrated into bins with the width of 0.004ppm using the AMIX package (v3.8, Bruker Biospin), and the region δ 4.75–4.93 for water peaks was removed. Each bin area was normalized to the dry weight of feces used for the extraction. For the urine spectra, the spectral region δ0.5–9.35 was integrated into bins with the width of 0.004ppm. The regions for water (δ4.71–5.10) and urea (δ5.43–6.20) peaks were removed, and each bin area was normalized to the total area of the respective spectrum. Some regions, including δ7.00–7.25, δ7.86–8.00 and δ8.05–8.20, were discarded after the normalization because of imperfect peak alignment.
Gut Microbiota Profiling
Global Structural Analysis of Gut Microbiota
Functional Annotation
Gut Microbiota Transplantation
Results
Discussion The obesity associated with PWS seems to be genetically determined, yet the primary drivers are still food craving and low calorie expenditure, similar to simple obesity. Energy restricted diets have been recommended for weight control in PWS children (Bonfig et al., 2009). Most of these diets do not improve satiety of PWS children, making it difficult for long-term adherence. Energy-restricted diet with low carbohydrates can lead to production of toxic metabolites by the gut bacteria, thus may be detrimental to health for long term use (Russell et al., 2011). In PWS children, a reduced energy diet with well balanced macronutrients and increased fiber intake yielded a better weight control than diets with just reduced energy intake (Miller et al., 2013). However, the impact of this type of diet on gut microbiota as a potential mechanism for its contribution to obesity improvement remains elusive in PWS children.