MENCARELLI ET AL. recommended the use of bootstrapping procedures when the theo- retical distribution of a statistic is complicated or unknown. As the bootstrapping procedure is distribution-independent, it provides an indirect method to assess the properties of the distribution underlying the sample and the parameters of interest. There was an obvious age and sex relation of the data. Subsequently the three male patients with the highest Cr/Crn ratio were selected. The ratio was consistently abnormal in two patients but two repeat samples in patient three showed normal results (Fig. 2; Table I). Clinical Description of the Patients With the Highest Cr/Crn Ratio Patient 1 (#1115) (Fig. 3a) is a 18-year-old boy, the third child of nonconsanguineous and healthy parents. His mother showed a mild ID. He was born at term by caesarian after a pregnancy of normal course. At birth he showed a weight of 3,030 g (25–50th centile) and hypotonia. Psychomotor delay was noted since early infancy: crawl- ing was acquired after 1 year and ﬁrst single words after 1 year. He had two episodes of seizures, one of which in fever. Karyotype with resolution of 400 bands and molecular analysis of FMR1, SCN1A, and GABRG2 genes were normal. Physical examination at 16 years and 8 months showed: height 174 cm (50–75th centile), weight 83 kg (90–97th centile), OFC 57 cm (90–97th centile), sparse eyebrows, long ears (7 cm, >þ2DS), M-shaped upper lip, open mouth, normal hand- and foot length. He was not able to formulate sentences and showed hyperactivity, hyperphagia, and aggressiveness. Patient 2 (#726) (Fig. 3b) is a 12-year-7 month-old boy, ﬁrst child of nonconsanguineous and healthy parents. He was born at term by caesarian after a pregnancy of normal course. His birth weight was 3,500 g (50th centile). He showed speech delay and three episodes of myoclonic seizures. Psychometric evaluation at 11 years recorded an IQ of 50. Array-CGH analysis (105 K) was normal. Physical exami- nation at 11 years and 6 months showed: height 138 cm (25th centile), weight 35 kg (50th centile), OFC 54 cm (75th centile), triangular face, and long ears. His language was simple and he showed hyperactivity and abnormal behavior. 2449 Brain MRS In Patients 1 and 2 the results of Cho/Cr and NAA/Cr measure- ments in the white matter of centrum ovale demonstrated a signiﬁcant abnormally high ratio compared to age matched healthy controls (Fig. 4). For Patient 1 the following ratios were found: Cho/Cr ¼ 4.2, NAA/Cr ¼ 12.8 at TE of 30 msec; Cho/Cr ¼ 4.15 and NAA/Cr ¼ 12.7 at TE of 135 msec. In Patient 2 these amounted to Cho/Cr ¼ 4.7 and NAA/Cr ¼ 9.3 at TE of 30 msec; Cho/Cr ¼ 4.2 and NAA/Cr ¼ 9.2 at TE of 135 msec. Patient 3 showed normal Cho/Cr and NAA/Cr ratios. In a control group (subjects, n ¼ 10) at TE of 30 msec, the mean of Cho/Cr is 1.00 and the SD is 0.26; the mean of NAA/Cr is 2.21 and the SD is 0.35. Molecular Analysis of SLC6A8 Gene SLC6A8 gene analysis revealed two missense mutations: in Patient 1 a novel mutation was identiﬁed (NM_005629.3:c.993C>G p.Asn331Lys) that was inherited from his mother who has a borderline IQ. Skewed X-inactivation was excluded. The mutation lies in a cytoplasmic domain and affects a residue which is highly conserved in different species (including Mus musculus BC141067, Rattus norvegicus NW_001084770, Bos taurus NM_174611, and Oryctolagus cuniculus NM_001082397). In Patient 2 a de novo mutation (NM_005629.3:c.1171C>T p.Arg391Trp, known mutation-PMID17465020: Rosenberg 2007) was identiﬁed. The mutation lies in a extracellular domain and affects a residue which is highly conserved in different species. Molecular analysis of SLC6A8 gene showed no mutation in Patient 3 (Fig. 3c). DISCUSSION Since its ﬁrst description in 2001, approximately 150 patients have been reported in the literature with a diagnosis of X-linked creatine transporter deﬁciency. The low number of reported cases is def- initely an underestimation of the frequency of this condition, since present data suggest that SLC6A8 mutations will have a 3–5 times greater frequency than most other X-linked ID genes [Clark et al., FIG. 3. Photographs of Patient 1 (a), Patient 2 (b) and Patient 3 (c).