2448 Molecular Analysis of the SLC6A8 Gene The 13 exons and the consensus splice sites of the SLC6A8 gene were analyzed. A total of eight PCR reactions were performed: exons 5-6-7, 8- 9-10, and 11-12, were sufﬁciently close to be ampliﬁed as three frag- ments. Primers and PCR conditions are available upon request. PCR products were puriﬁed with ExoSAP-IT (USB Corporation, Cleveland, OH) and bidirectionally sequenced on an ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA) using the same PCR primers. Brain MRI and 1H MRS Patients underwent 1.5 T magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (1H MRS) examinations. A multi- voxel technique with SE sequences at TE 30 msec and TE 135 msec was used. The multivoxels 1H MRS 2D technique was acquired on the centrum ovale. All voxels examined in the patients had normal appearing brain tissue on conventional MRI examination. The local magnetic ﬁeld homogeneity is automatically optimized with an auto- shim procedure and adjusted for chemical-shift-water suppression. 1H-MRS raw data were transferred to a Leonardo Workstation, recon- structed and processed by Siemens software (Erlangen, Deutschland). Peak areas of choline (Cho), creatine (Cr), and N-acetyl aspartate (NAA) were estimated by numeric integration. Metabolite signals were measured from single spectra. White matter intravoxel (Cr/Cho, Cho/Cr, and NAA/Cr) ratios were calculated from the peak areas. RESULTS Urine Proton NMR Spectroscopy As no age- and sex-related reference values are available for the Cr/Crn ratio in urine we derived reference intervals (p5, 50, and 95) from our AMERICAN JOURNAL OF MEDICAL GENETICS PART A total patient group. The data did not show a Gaussian distribution and this was also not the case after logarithmic transformation. To obtain the reference intervals shown in Table I a bootstrap resampling procedure was used for data of which the distribution is not known. The dataset was split in age group classes (0–5, 6–10, 11–15, 16–20, and >20 years) (Fig. 1). Within each class we performed bootstrap resampling (n ¼ 1,000). Bootstrap statistical techniques are computer intensive methods falling in the resampling techniques used as an alternative based parametric statistic (p < 0,05). Ader et al. [2008] FIG. 1. Median, 9th and 99th centile for each age classes as a result of the above-described bootstrap technique starting from the Cr/Crn ratios of 1,347 urine samples. FIG. 2. Proton NMR spectra measured at pH 2.50. a: Standard 0.1 M solution of pure substances creatine (3.05 and 4.11 ppm) and creatinine (3.13 and 4.29 ppm). We can clearly see the separation between the signals: a singlet at 3.05 ppm from the methyl of creatine and a singlet at 3.13 ppm from the methyl of creatinine. The two other singlets come from the methylene of creatine (at 4.11 ppm) and creatinine (at 4.29 ppm). b: Urine spectrum of a healthy subject. c: Urine spectrum of a patient affected by creatine transporter defect. The arrows indicate the creatine and creatinine signals.