In the present study we have identified 16 new patients diagnosed with a PCD over a period of 28 months, as well as three affected siblings. Intellectual disability and attention deficit were, along with motor and speech delays, the main symptoms reported by the medical staff involved in the diagnosis of these disorders. Nevertheless, it is surprising that intellectual disability, which was a sign observed by the medical examiners in all patients with PCD, was not the main cause leading the parents to consult. This might reflect the difficulty of identifying intellectual disability in the very young child or in turn to of associating intellectual disability with PCD. These disorders are known to progress via irreversible lesions of the brain, so therapeutic measures are needed to be taken early in the life of the patients. For GAMT patients, supplementation with creatine together with a GAA lowering strategy (ornithine supplementation with or without arginine restriction) may represent an efficient treatment strategy [43–46] and may provide some medical neurological benefit for GAMT patients. In these conditions, patients may recover a normal brain creatine peak as detected by 1H-MRS as well as normal urine GAA levels. They also improve clinically with better language and social development and apparently some regression of the epilepsy . By contrast, in patients with SLC6A8 deficiency, supplementations with creatine and its precursors arginine or glycine, though improving muscular signs, were found to be without substantial benefit on cognitive and psychiatric signs, and failed to modify the creatine signal in 1H-MRS imaging of the brain , suggesting the inefficacy of these supplementations in improving brain as stated by other studies [4, 19, 30, 49–52]. Though there is hope to treat efficiently this group of patients through the discovery of creatine pro-drugs capable of entering the brain and cells via a by-pass of the SCL6A8 creatine transporter, some clinical studies also question the inefficacy of the therapeutic measures mentioned above and, on the contrary, show beneficial effects with creatine and/or arginine supplementations in patients with SLC6A8 deficiency. In a heterozygous female patient with intractable epilepsy, treatment with creatine combined with arginine and glycine completely resolved seizures . Creatine supplementation was also described to improve the neurological, language and behavioral status and was associated with a rise in the brain creatine peak as demonstrated by MRS in a child with SLC6A8 deficiency . In a recent study, creatine deficient patients were also shown to be improved by a L-arginine-based therapy which positively impacted daily living skills, lowered the frequency of epileptic episodes and induced a mild increase in brain creatine and phosphocreatine MRS signals although normal cerebral levels of these metabolites were not recovered .
The calculated prevalence of PCD in our cohort was 0.25%. This result was not expected because most studies report a higher prevalence of PCD, notably SLC6A8 deficiency, which is generally estimated between 1% and 3% of the population affected with intellectual disability [56–59]. However, this prevalence has been considered to be an overestimation of the real prevalence of PCD. Because PCDs are monogenic disorders, their prevalence was proposed to be no different from that of other nonsyndromic diseases such ARX (Aristaless-Related homeobox gene located on X chromosome) and therefore closer to 0.1% - 0.3% . Thus, for the first time, our screening study provides strong practice-based evidence confirming this estimated prevalence for PCD. The girl diagnosed with SLC6A8 deficiency presented a severe phenotype similar to affected male patients, an observation that is not surprising in view of recent work [34, 61, 62] also describing this X-linked disorder in the female population with intellectual disability. In this respect, screening for SLC6A8 deficiency in female patients should be included in the diagnostic workup since this disorder still remains under-diagnosed. However, it should be noted that if the urinary U-CT/CTN ratio is not increased the diagnosis cannot be ruled out because it has been shown that the majority of female patients with a heterozygous mutation in SLC6A8 have a normal ratio, although the average ratio of this group is increased . It might be recommended to perform brain MRS and/or SLC6A8 gene studies in female patients with suspected creatine transporter deficiency.
Five mutations were identified for GAMT gene in 6 unrelated families. These included 3 new mutations, c.289C > T, c.391 + 15G > T and c.577C > T, and two previously described mutations, c.299_311dup13  and c.506G > A  (Table 2). The new mutation c391 + 15G > T was considered pathogenic by the creation of an alternative splicing donor site in intron 3 (Alamut software), by the segregation of this mutation in his parents and because it was linked with decreased GAMT activity in cultured fibroblasts.
The SLC6A8 gene was analyzed in 11 patients from 9 unrelated families. Nine mutations were identified throughout the gene, including 4 new mutations (2 missense (c.1208C > A and c.926C > A), 1 frameshift (c.930delG) and 1 splicing (c.1393-1G > A) mutation), and 5 previously described mutations (c.321_323delCTT , c.942_944delCTT , c.1006_1008delAAC , c.778-2A > G and c.1519_1543del ) (Table 3). In accordance with the results of Clark et al. , we found a fairly high proportion of frameshift and splicing mutations in our patients, and approximately one fifth of the mutations of SLC6A8, but not GAMT, were attributed to neo-mutation, germinal or somatic mosaicism events.
Interestingly, DNA studies performed in our patients clearly showed that mutations were essentially patient/family specific and highlighted the absence of hot spot mutations in these genes. Finally, as a mutation was identified in each patient for whom DNA was analyzed, the study of the target gene appears to be a key diagnostic step for PCD in suspect patients, keeping in mind that secondary creatine disorders such as deficiencies of ornithine delta-aminotransferase (EC 184.108.40.206) [66, 67], urea [68–71] and homocysteine/methionine [72, 73] cycles, and succinate semialdehyde dehydrogenase (SSADH) (EC 220.127.116.11) [74, 75] may also be alternative causes of abnormal laboratory values of creatine metabolism markers.