MENCARELLI ET AL. 2006]. The frequency of SLC6A8 mutations seems to be comparable to that of FMR1 mutations and thus SLC6A8 may be considered a major contributor to ID in the male population [Clark et al., 2006]. The few diagnosed patients reveal that this condition is not recognizable from a clinical point of view. Aspeciﬁc clinical phe- notypes also occur in other X-linked ID-related diseases. However, creatine levels in biological ﬂuids represent a strong diagnostic hint for this condition and this metabolite should be investigated in order to improve the diagnostic yield. Brain MRS, demonstrating creatine depletion, is the golden standard for the diagnosis. How- ever, it is an expensive analysis not available in many hospitals and it is an invasive diagnostic tool due to the need of sedation. It thus cannot be considered a ﬁrst level-screening test. Therefore, it would be important to use a rapid and less expensive test to select patients for further diagnostic analyses. Urine screening has been employed in at least three studies using gas chromatography/mass spectrometry or LC/mass spectrometry. In 2007, Arias et al. [2007] determined Cr/Crn ratio in urine samples from 1,600 male patients with ID and/or autism and identiﬁed 4 true positive cases (0.3%) and 21 false positive cases (1.8%). More recently, in a study by Puusepp et al. [2009] 49 ID familial cases were preliminary selected for gene analysis by urine creatine assessment. In this study, the biochemical testing showed 18% false positives. In the study of Mercimeck-Mahmutoglu et al. [2009] the Cr/Crn ratio was determined by tandem mass- spectrometry leading to the identiﬁcation of 2 true positive cases out of 29 individuals (6.9% false positives). In the present work we applied a biochemical screening test, by proton NMR spectroscopy measuring urine samples from 258 mostly sporadic ID males. We analyzed the Cr/Crn ratio followed by SLC6A8 gene analysis in the three patients with the highest ratio. In two patients a patho- genic mutation was found conﬁrming the suspected creatine trans- porter defect. The increased ratio in the third patient was most likely caused by dietary interference. This illustrates that a single urine sample is not enough to be sure of this diagnosis, so we recommend to attest an increased Cr/Crn ratio in at least two samples of a patient prior to more expensive and invasive testing. Genetic analysis is advised when the Cr/Crn ratio is consistently abnormal in almost two samples. It has the advantage to estimate the recurrence risk of the deﬁcit for all family members. Our work demonstrates that urine NMR spectroscopy can be used to identify patients with a creatine transporter defect. It has the advantage that only little sample-pretreatment is needed (the pH adjustment). NMR spectroscopy will be also able to ﬁnd cases with GAMT deﬁciency (guanidinoacetate methyltransferase) [Tassini et al., 2010]. The analysis can also be performed in frozen samples, which can be collected in peripheral hospitals and sent to referral hospitals later. We have found a consistently abnormal Cr/Crn urine ratio in two patients and conﬁrmed the decreased creatine concentration in brain of these patients. Results presented here and those previously published by Clarke et al. [2006] stress that a signiﬁcant number of males with ID may have an SLC6A8 mutation. Even though in our series of familial cases we did not identify any positive patient, present data suggest that the prevalence of SLC6A8 mutations is even higher in familial cases reaching a 2% [Rosenberg et al., 2004; Puusepp et al., 2009]. 2451 An a posteriori re-evaluation of the clinical phenotype of positive patients conﬁrmed that it is not speciﬁc. Both patients in whom we detect a SLC6A8 mutation present moderate ID, mostly related to speech and language, hyperactivity, behavioral disturbances, and no dysmorphic features. Therefore, we suggest to perform urine NMR as a ﬁrst level-screening test in males with ID regardless of the family history and to proceed to SCL6A8 molecular analysis, if a high Cr/Crn ratio is consistent. ACKNOWLEDGMENTS We would like to thank our patients and their parents for their precious collaboration. This work has been supported by ‘Cell Lines and DNA Bank of Rett syndrome, X mental retardation and other genetic diseases’ (Meby ‘Cell Lines and DNA Bank of Rett syndrome, X mental retardation and other genetic diseases’ (Medical Genetics-Siena) - Telethon Genetic Biobank Network (Project No. GTB07001C to AR) and Telethon grant GDP09117 to AR.dical Genetics-Siena) - Telethon Genetic Biobank Network (Project No. GTB07001C to AR) and Telethon grant GP06170 to AR. 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