MENCARELLI ET AL. In 2004, the same group assessed the frequency of this condition in a group of 288 males with an X-linked pedigree with at least two affected males [Rosenberg et al., 2004]. By gene sequence analysis, they found that SLC6A8 mutations account for 2% of X-linked ID males (6/288). In 2006, the same group investigated the frequency of this condition among ID sporadic cases [Clark et al., 2006]. In a total of 478 ID males by gene sequence analysis they found 4 pathogenic mutations concluding that the condition may have at least a frequency of 0.8% among ID males. Afterwards, a lower prevalence (0.3%) was observed in a cohort of 1,600 patients with ID of unknown origin, previously selected by HPLC–MS/MS [Arias et al., 2007]. In 2009 an Estonian group explored the frequency of the condition in 49 familial ID case with a compatible X-linked transmission by measuring the creatine/creatinine (Cr/Crn) ratio in urine [Puusepp et al., 2009]. The authors found eight false positive families and one SLC6A8 mutated family con- ﬁrming the frequency of 2% among X-linked ID families. Mercimeck-Mahmutoglu et al. [2009] developed a stable isotope dilution tandem mass-spectrometry method to determine the Cr/Crn ratio in 975 individuals. They conﬁrmed a SLC6A8 mutation in 2 males among 87 with one or more symptoms at risk for SLC6A8 defects, giving a frequency of SLC6A8 deﬁciency of 2.3% in the cohort at risk. Since the ﬁrst description, more than 60 index cases affected by this disorder and a total of 150 patients have been ascertained [Sto€ckler-Ipsiroglu and Salomons, 2006]. The clinical phenotype ranges from mild ID and speech delay to severe ID, seizures, and behavioral disorders. The diagnosis cannot be established on clinical grounds only as the clinical signs and symptoms are rather aspeciﬁc and the suspicion of the disease is based on the assessment of brain creatine level by MRS or by measurement of the Cr/Crn ratio in urine [Almeida et al., 2004]. We have explored the feasibility of NMR spectroscopy of the urine to this end and included 258 male urine samples in our study. In this study 258 urine NMR spectra of male children were studied. We selected three male patients on the basis of their Cr/Crn ratio and found that two have a pathogenic molecular defect in creatine transporter gene SLC6A8. 2447 MATERIALS AND METHODS Patients A cohort of 258 males with ID was included in the medical study. Among these, 231 were sporadic cases, while 27 had at least one affected brother. All patients were evaluated by a clinical geneticist (AR, MAM, or MP) and clinically recognizable syndromes were excluded. In all cases molecular analysis of FMR1 gene resulted normal. Urine NMR Analysis Urine 1H-NMR measurements were carried out on a 600 MHz Bruker DRX Avance Spectrometer with a Selective Inverse Probe (SEI) equipped with Z gradient coil. Spectra were acquired at the constant temperature of 298.0 0.1 K using a 90 pulse. A 10 sec delay was included in the pulse sequence to allow T1 relaxation as previously described [Engelke et al., 2009]. Suppression of the water signal was achieved by applying a saturation pulse of 2 sec duration at the water resonance. 32K data points per scan were used and 128 transients were accumulated. The total experimental time for each spectrum was 27 min. Each urine sample was measured after cen- trifugation at 2,000 rpm for 5 min. Sample (550 ml) plus 50 ml of a TSP-d4 20 mM solution, were transferred into a 0.5 mm (outer diameter) NMR tube at pH 2.50 0.02. The chemical shift of ionizable substances is highly dependent on pH. At pH 2.50 all chemical shift values are reproducible within 0.01 ppm [Lehnert and Hunkler, 1986]. Moreover, under these conditions, the methyl signals of creatine and creatinine are clearly separated (3.05 ppm for the methyl signal of creatine and 3.13 ppm for the methyl signal of creatinine). The pH adjustment to 2.50 was the only sample pretreat- ment. The pH was adjusted with a minimal volume of HCl, starting from a 3 M and ending with a 0.05 M solution (dilution effect less than 5%). The pH meter used was model 420A Orion connected with a MI-412 combination electrode from Microelectrodes, Inc., Bed- ford, New Hampshire. The Cr/Crn ratio could be measured directly from the areas of the signals without any additional quantiﬁcation. Statistical calculations using a bootstrap resampling procedure have been carried out using ‘‘R’’, a free software environment for statistical computing and graphics (R Development Core Team, 2010). TABLE I. Creatine/Creatinine Ratios Measured by NMR Urine Spectroscopy in Male Patients, Related SLC6A8 Gene Mutations and Age Classes With the Related 5, 50, 95 Percentiles as Calculated After Bootstrap Resampling Patient 1 2 3 NMR Cr/Crn urine ratio 2.080 3.350 2.500 2.300 2.090 2.120 1.880 1.590 3.360 0.640 0.120 Age at the time of sample collection (years) 14.0 15.5 16.5 17.5 7.0 7.5 8.5 12.5 5.0 10.0 10.0 Age classes (years) 11–15 11–15 16–20 16–20 6–10 6–10 6–10 11–15 0–5 6–10 6–10 p5 0.008 0.008 0.004 0.004 0.008 0.008 0.008 0.008 0.010 0.008 0.008 p50 0.096 0.096 0.016 0.016 0.214 0.214 0.214 0.096 0.333 0.214 0.214 p95 0.648 0.648 0.275 0.275 0.870 0.870 0.870 0.648 1.155 0.870 0.870 SLC6A8 mutation c.C993G p.N331K c.C1171T p.R391W No mutations