Targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) employing an enzymatic assay for γ-hydroxybutyric acid (GHB) in biofluids.

HYPOTHESIS: An enzymatic assay for quantification of γ-hydroxybutyric acid (GHB) in biofluids can be employed for targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) in selected populations. RATIONALE: We used a two-tiered study approach, in which the first study (proof of concept) examined 7 urine samples derived from patients with SSADHD and 5 controls, and the second study (feasibility study) examined a broader sample population of patients and controls, including plasma. OBJECTIVE: Split samples of urine and plasma (anonymized) were evaluated by enzymatic assay, gas chromatography alone (proof of concept) and gas chromatography-mass spectrometry, and the results compared. METHOD: Multiple detection methods have been developed to detect GHB. We evaluated an enzymatic assay which employs recombinant GHB dehydrogenase coupled to NADH production, the latter quantified on a Cobas Integra 400 Plus. Results: In our proof of concept study, we analyzed 12 urine samples (5 controls, 7 SSADHD), and in the feasibility study we evaluated 33 urine samples (23 controls, 10 SSADHD) and 31 plasma samples (14 controls, 17 SSADHD). The enzymatic assay carried out on a routine clinical chemistry analyzer was robust, revealing excellent agreement with instrumental methods in urine (GC-FID: r = 0.997, p ≤ 0.001; GC-MS: r = 0.99, p ≤ 0.001); however, the assay slightly over-estimated GHB levels in plasma, especially those in which GHB levels were low. Conversely, correlations for the enzymatic assay with comparator methods for higher plasma GHB levels were excellent (GC-MS; r = 0.993, p ≤ 0.001). CONCLUSION: We have evaluated the capacity of this enzymatic assay to identify patients with SSADHD via quantitation of GHB. The data suggests that the enzymatic assay may be a suitable screening method to detect SSADHD in selected populations using urine. In addition, the assay can be used in basic research the elucidate the mechanism of the underlying disease or monitor GHB- levels for the evaluation of drug candidates. SYNOPSIS: An enzymatic assay for GHB in biofluids was evaluated as a screening method for SSADHD and found to be reliable in urine, but in need of refinement for application to plasma.

Therapeutic relevance of mTOR inhibition in murine succinate semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism.

Aldehyde dehydrogenase 5a1-deficient (aldh5a1-/-) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts autophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypothesis that aldh5a1-/- mice would show altered levels of mRNA for genes associated with mTOR signaling and oxidative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1-/- mice. These metabolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1-/- mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2-nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1-/- mice, and several were significantly improved with the administration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxidative stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD.

Correlation of blood biomarkers with age informs pathomechanisms in succinic semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism.

We hypothesized that blood levels of γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB), biomarkers of succinic semialdehyde dehydrogenase deficiency (SSADHD), would correlate with age. GABA and GHB were quantified in plasma and red blood cells (RBCs) from 18 patients (age range 5-41 years; median 8). Both metabolites negatively correlated with age (P < 0.05). Plasma and RBC GHB declined with age, reaching a nadir and approximate steady state by 10 years. Declining plasma GABA achieved this approximate steady state at 30-40 years of age. These biomarker relationships may reflect further GABA- and GHB-ergic neurotransmission imbalances that correlate with the onset of adolescent/adulthood neuropsychiatric morbidity and epilepsy in SSADHD.

Pyridoxine responsive epilepsy caused by a novel homozygous PNPO mutation.

We report a patient with anti-epileptic treatment refractory neonatal seizures responsive to pyridoxine. Biochemical analysis revealed normal markers for antiquitin deficiency and also mutation analysis of the ALDH7A1 (Antiquitin) gene was negative. Mutation analysis of the PNPO gene revealed a novel, homozygous, presumed pathogenic mutation (c.481C > T; p.(Arg161Cys)). Measurements of B6 vitamers in a CSF sample after pyridoxine administration revealed elevated pyridoxamine as the only metabolic marker for PNPO deficiency. With pyridoxine monotherapy the patient is seizure free and neurodevelopmental outcome at the age of 14 months is normal.

A pilot study to estimate incidence of guanidinoacetate methyltransferase deficiency in newborns by direct sequencing of the GAMT gene.

BACKGROUND: GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis causing developmental delays or intellectual disability in untreated patients as a result of irreversible brain damage occurring prior to diagnosis. Normal neurodevelopmental outcome has been reported in patients treated from neonatal period highlighting the importance of early treatment. METHODS: Five hundred anonymized newborns from the National Newborn Screening Program of The Netherlands were included into this pilot study. Direct sequencing of the coding region of the GAMT gene was applied following DNA extraction. The disease causing nature of novel missense variants in the GAMT gene was studied by overexpression studies. GAA and creatine was measured in blood dot spots. RESULTS: We detected two carriers, one with a known common (c.327G>A) and one with a novel mutation (c.297_309dup (p.Arg105Glyfs*) in the GAMT gene. The estimated incidence of GAMT deficiency was 1:250,000. We also detected five novel missense variants. Overexpression of these variants in GAMT deficient fibroblasts did restore GAMT activity and thus all were considered rare, but not disease causing variants including the c.131G>T (p.Arg44Leu) variant. Interestingly, this variant was predicted to be pathogenic by in silico analysis. The variants were included in the Leiden Open Variation Database (LOVD) database (www.LOVD.nl/GAMT). The average GAA level was 1.14µmol/L±0.45 standard deviations. The average creatine level was 408µmol/L±106. The average GAA/creatine ratio was 2.94±0.136. CONCLUSION: The estimated incidence of GAMT deficiency is 1:250,000 newborns based on our pilot study. The newborn screening for GAMT deficiency should be implemented to identify patients at the asymptomatic stage to achieve normal neurodevelopmental outcome for this treatable neurometabolic disease. Biochemical investigations including GAA, creatine and GAMT enzyme activity measurements are essential to confirm the diagnosis of GAMT deficiency. According to availability, all missense variants can be assessed functionally, as in silico prediction analysis of missense variants is not sufficient to confirm the pathogenicity of missense variants.

Genotype-phenotype correlation of contiguous gene deletions of SLC6A8, BCAP31 and ABCD1.

The BCAP31 gene is located between SLC6A8, associated with X-linked creatine transporter deficiency, and ABCD1, associated with X-linked adrenoleukodystrophy. Recently, loss-of-function mutations in BCAP31 were reported in association with severe developmental delay, deafness and dystonia. We characterized the break points in eight patients with deletions of SLC6A8, BCAP31 and/or ABCD1 and studied the genotype-phenotype correlations. The phenotype in patients with contiguous gene deletions involving BCAP31 overlaps with the phenotype of isolated BCAP31 deficiency. Only deletions involving both BCAP31 and ABCD1 were associated with hepatic cholestasis and death before 1 year, which might be explained by a synergistic effect. Remarkably, a patient with an isolated deletion at the 3'-end of SLC6A8 had a similar severe phenotype as seen in BCAP31 deficiency but without deafness. This might be caused by the disturbance of a regulatory element between SLC6A8 and BCAP31.

[L-2-hydroxyglutaric aciduria: report on two cases]. / Acidurie L-2-hydroxyglutarique : à propos de 2 cas.

L-2-hydroxyglutaric aciduria is a rare genetic neurometabolic disease. It occurs in childhood with mental retardation, cerebellar ataxia, and epilepsy. Macrocephaly is present in half of the cases. Diagnosis is based on clinical symptoms, biological and radiological findings, and molecular testing. Specific treatments can improve the spontaneous progression of the disease. We examined two independent patients who presented with L-2-hydroxyglutaric aciduria. Clinical presentation led to cerebral MRI and urinary organic acid chromatography. The genetic analysis confirmed the diagnosis. Under specific treatment, the progression of the disease was subsequently stopped. L-2-hydroxyglutaric aciduria shares common symptoms with other genetic and metabolic diseases. However, the association of a distinct phenotype and typical MRI abnormalities (such as a high signal in the subcortical white matter, pallidum, and dentate nuclei) should draw the clinician's attention to this diagnosis. It can easily be suspected with a simple urinary analysis and can then be confirmed by genetic testing. With this case report, we show the importance of genetic identification to begin treatment with riboflavin. Early detection of L-2-hydroxyglutaric aciduria based on MRI abnormalities can enable rapid initiation of treatment and prevent disease progression.

Creatine deficiency syndrome. A treatable myopathy due to arginine-glycine amidinotransferase (AGAT) deficiency.

We report two sisters, aged 11 and 6years, with AGAT deficiency syndrome (OMIM 612718) which is the least common creatine deficiency syndrome. They were born full-term to consanguineous parents and had moderate developmental delay. Examination showed an important language delay, a progressive proximal muscular weakness in the lower limbs with Gowers sign and myopathic electromyography. Investigations revealed undetectable guanidinoacetate and low level of creatine in plasma and urine, characteristic findings of AGAT deficiency syndrome. Brain magnetic resonance spectroscopy showed a markedly reduced level of creatine. Guanidinoacetate methyltransferase (GATM) gene sequencing revealed a homozygous missense mutation in exon 4:c.608A>C, (p.Tyr203Ser). Thirteen months after beginning the treatment with oral creatine monohydrate 200mg/kg/day, then 400mg/kg/day, there was a dramatic improvement in muscle strength with Gowers sign disappearance in both patients, and a mild improvement in language and cognitive functions. AGAT deficiency syndrome should be considered in all patients with language retardation and cognitive impairment associated to a myopathy of unknown etiology such that early diagnosis must lead to creatine supplementation to cure the myopathy and improve language and cognitive function.

Guanidinoacetate methyltransferase deficiency: first steps to newborn screening for a treatable neurometabolic disease.

BACKGROUND: GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis resulting in severe neurological complications in untreated patients. Currently available treatment is only successful to stop disease progression, but is not sufficient to reverse neurological complications occurring prior to diagnosis. Normal neurodevelopmental outcome in a patient, treated in the newborn period, highlights the importance of early diagnosis. METHODS: Targeted mutation analysis (c.59G>C and c.327G>A) in the GAMT gene by the QIAxcel system and GAA measurement by a novel two-tier method were performed in 3000 anonymized newborn blood dot spot cards. RESULTS: None of the targeted mutations were detected in any newborn. Two novel heterozygous variants (c.283_285dupGTC; p.Val95dup and c.278_283delinsCTCGATGCAC; p.Asp93AlafsX35) were identified by coincidence. Carrier frequency for these insertion/deletion types of GAMT mutations was 1/1475 in this small cohort of newborns. GAA levels were at or above the 99th percentile (3.12 µmol/l) in 4 newborns. Second-tier testing showed normal results for 4 newborns revealing 0.1% false positive rate. No GAMT mutations were identified in 4 of the newborns with elevated GAA levels in the first tier testing. CONCLUSION: This is the first two-tier study to investigate carrier frequency of GAMT deficiency in the small cohort of newborn population to establish evidence base for the first steps toward newborn screening for this treatable neurometabolic disorder.

Efficacy of vigabatrin intervention in a mild phenotypic expression of succinic semialdehyde dehydrogenase deficiency.

We report a patient with succinic semialdehyde dehydrogenase deficiency who presented a mild phenotype including developmental language delay, in association with the typical elevations of 4-hydroxybutyric acid (GHB) in biological fluids and MRI alterations. Two pathogenic mutations were identified one transversion (c.278 G>T) in exon 1 and another (c.1557 T>G) in exon 10. Both parents are carriers of one of the mutations, confirming compound-heterozygosity in their affected child. To reduce the GHB levels in body fluids, a treatment with vigabatrin at low dose (25 mg/kg per day) was started, monitoring its efficacy by clinical and neurochemical follow-up. After 9 months of therapy with vigabatrin, a significant reduction of GHB concentrations in urine and CSF was observed; after 36 months, a significant improvement of communicative skills, not previously reported, was referred. These results support the hypothesis that the clinical improvement is correlated to the reduction in the GHB levels and the importance of considering the SSADH deficiency in the differential diagnosis of patients with mental retardation and language delay.

Poster - Thur Eve - 18: Characterization of a camera and LED lightbox imaging system for radiochromic film dosimetry.

Radiation therapy treatment modalities continue to develop and have become increasingly complex. With this, dose verification and quality assurance (QA) is of great importance to ensure that a prescribed dose is accurately and precisely delivered to a patient. Radiochromic film dosimetry has been adopted as a convenient option for QA, because it is relatively energy independent, is near tissue equivalent, and has high spatial resolution. Unfortunately, it is not always easy to use. In this study, preliminary work towards developing a novel method of imaging radiochromic film is presented. The setup consists of a camera mounted vertically above a lightbox containing red LEDs, interfaced with computer image acquisition software. Imaging results from this system will be compared with imaging performed using an Epson Expression 10000XL scanner (a device in common clinical use). The lightbox imaging technique with camera readout is much faster relative to a flatbed scanner. The film measurements made using the camera are independent of film orientation, and show reduced artifacts, so that there are fewer corrections required compared to the use of flatbed scanners. Optical scatter also appears to be less of an issue with this design than with the flat bed scanner. While further work needs to be done to optimize the lightbox imaging system, the lightbox system shows great promise for a rapid, simple, and orientation independent setup, improving on existing film scanning systems.

Poster - Thur Eve - 29: Detecting changes in IMRT QA using statistical process control.

Statistical process control (SPC) methods were used to analyze 239 measurement based individual IMRT QA events. The selected IMRT QA events were all head and neck (H&N) cases with 70Gy in 35 fractions, and all prostate cases with 76Gy in 38 fractions planned between March 2009 and 2012. The results were used to determine if the tolerance limits currently being used for IMRT QA were able to indicate if the process was under control. The SPC calculations were repeated for IMRT QA of the same type of cases that were planned after the treatment planning system was upgraded from Eclipse version 8.1.18 to version 10.0.39. The initial tolerance limits were found to be acceptable for two of the three metrics tested prior to the upgrade. After the upgrade to the treatment planning system the SPC analysis found that the a priori limits were no longer capable of indicating control for 2 of the 3 metrics analyzed. The changes in the IMRT QA results were clearly identified using SPC, indicating that it is a useful tool for finding changes in the IMRT QA process. Routine application of SPC to IMRT QA results would help to distinguish unintentional trends and changes from the random variation in the IMRT QA results for individual plans.

A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: novel avenues for biochemical and therapeutic studies.

The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg(140) to Gln(140) or Gly(140) (IDH2(wt/R140Q), IDH2(wt/R140G)) in d-2-hydroxyglutaric aciduria (D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1(R132H) and IDH2(R172K) mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate (D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2(wt/R140Q) gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2(wt/R140Q) enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4-(2)H(4)]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2(wt/R140Q) enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4nmolh(-1)mgprotein(-1) vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2(wt/R140Q) activity was obtained with oxaloacetate, which competitively inhibited IDH2(wt/R140Q) activity. Lymphoblast IDH2(wt/R140Q) showed long-term cell culture stability without loss of the heterozygous IDH2(wt/R140Q) mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.

Novel mutations in pyridoxine-dependent epilepsy.

PURPOSE: Pyridoxine-Dependent Epilepsy (PDE) is a rare autosomal recessive disease with neonatal seizures resistant to conventional anti-epileptic drugs. This metabolic disease has to be diagnosed early and treated to improve outcome. We report on two new mutations that open new prenatal prospects and suggest a new diagnostic procedure. CASE REPORT: We describe PDE in a neonate carrying two novel mutations in the ALDH7A1 gene: c.[852_856delCTTAG] + [1230C > A]; p.[(Phe410Leu)] + p.[(Leu285CysfsX26)]. This case also illustrates that diagnosis could have been made without any pyridoxine withdrawal, thanks to the measurement of biomarkers. The patient was successfully treated with pyridoxine supplementation and currently shows normal neurological development.

Clinical features and X-inactivation in females heterozygous for creatine transporter defect.

The creatine transporter defect is an X-linked cause of mental retardation. We investigated the clinical features and pattern of X-inactivation in a Dutch cohort of eight female heterozygotes. We show that symptoms of the creatine transporter defect (mental retardation, learning difficulties, and constipation) can be present in female heterozygotes. We further show that the diagnosis in females is not straightforward: (i) The creatine/creatinine ratio in urine was elevated only in three of eight females. (ii) Although as a group the females had a significantly decreased cerebral creatine concentration, individual females had creatine concentrations overlapping with normal controls. (iii) Skewed X-inactivation was found in the cultured fibroblasts, in favour of either the mutated or the wild-type allele, leading to either deficient or normal results in the creatine uptake studies in fibroblasts. Thus, screening by these tests is unreliable for the diagnosis. In addition, we found no consistent skewing of the X-inactivation in peripheral tissues indicating that there is no selection against the creatine transporter defect. We conclude that testing for creatine transporter defect should be considered in females with (mild) mental retardation. Screening by DNA analysis of the SLC6A8 gene is recommended.

Use of a long-chain triglyceride-restricted/medium-chain triglyceride-supplemented diet in a case of malonyl-CoA decarboxylase deficiency with cardiomyopathy.

Malonyl coenzyme A (CoA) decarboxylase (EC 4.1.1.9, MCD) deficiency, or malonic aciduria, is a rare inborn error of metabolism characterised by a variable phenotype of developmental delay, seizures, cardiomyopathy and acidosis. There is no consensus for dietary treatment in this condition. This case describes the effect of a long-chain triglyceride (LCT)-restricted/medium-chain triglyceride (MCT)-supplemented diet upon the progress of an affected child. A full-term Asian girl of birth weight 3590 g was screened for malonic aciduria after birth due to a positive family history. She had elevated urine malonic and methylmalonic acids and was presumably homozygous for a deleterious mutation in the MLYCD gene. Her echocardiography showed mild cardiomyopathy at 0.5 months of age, but heart function was good. She was treated with carnitine 100 mg/kg per day and continued a high-energy formula feed, as her growth was slow. At 3 months of age, echocardiography showed deteriorating cardiac function with a fractional shortening of 18%. She started an angiotensin-converting enzyme (ACE) inhibitor (Captopril). Over the next few months, her diet was altered to comprise 1.9% energy from LCT, 25% from MCT and the remainder carbohydrate. Cardiac function improved and was optimal at 23 months of age, with a fractional shortening of 28% and good systolic function. During a period of low MCT intake, her cardiac function was noted to deteriorate. This reversed and stabilised following reinstatement of the diet. This case of malonic aciduria with cardiomyopathy demonstrates improvement in cardiac function attributable to LCT-restricted/MCT-supplemented diet.

Déficit de succínico semialdehído deshidrogenasa. Disminución de los niveles de 4 OH butírico con dosis bajas de vigabatrina / Succinic semialdehyde dehydrogenase deficiency: Decrease in 4-OH-butyric acid levels with low doses of vigabatrin

La deficiencia de succínico semialdehído deshidrogenasa o aciduria gammahidroxibutírica (GHB) es una rara enfermedad autosómica recesiva por alteración en el metabolismo del neurotransmisor inhibidor del ácido gammaaminobutírico. Debido a la deficiencia de esta enzima, se produce acúmulo del metabolito ácido GHB. El espectro clínico es heterogéneo con distintas manifestaciones neurológicas. El tratamiento más utilizado es la vigabatrina, aunque se discute su eficacia. Presentamos el caso de una familia con 2 hijos afectados. El mayor presenta retraso en la adquisición de hitos motores, marcha liberada a los 2 años, dificultad en la psicomotricidad fina, hipotonía axial, normorreflexia y retraso en el lenguaje tanto comprensivo como expresivo. Se evalúa al menor por hipotonía axial. Estudio metabólico: aumento de ácido GHB en orina y en plasma. El diagnóstico se confirma mediante el análisis de mutaciones del gen ALDH5A1. Se trata con vigabatrina en dosis bajas, lo que conduce a una disminución de los niveles de GHB en plasma en más de dos tercios y una evolución clínica favorable (AU)

Succinic semialdehyde dehydrogenase deficiency (gamma-hydroxybutyric aciduria) is a rare neurometabolic disease caused by a deficiency in gamma-aminobutyric degradation, resulting in an increase in gamma-hydroxybutyric acid in biological fluids. The clinical spectrum is heterogeneous, including a variety of neurological manifestations and psychiatric symptoms. The treatment usually used is vigabatrin, but its clinical efficacy is under discussion. We present two affected siblings. The older brother was examined when he was 2.5 years old due to psychomotor and developmental delay, disturbances in motor coordination, axial hypotonia and language disability. His younger brother had mild axial hypotonia when 5 months old. Metabolic studies demonstrated a high plasma and urine concentration of gamma-hydroxybutyric acid. Mutation analysis of the gene ALDH5A1 confirmed the disease. After 1 year of treatment with low-doses of vigabatrin of the older patient, a decrease in gamma-hydroxybutyric acid plasma levels and a slow clinical improvement were observed (AU)

The screening of SLC6A8 deficiency among Estonian families with X-linked mental retardation.

The urinary creatine:creatinine (Cr:Crn) ratio was measured in males from 49 families with a family history compatible with X-linked mental retardation (XLMR) in order to estimate the prevalence of SLC6A8 deficiency in Estonia. We identified 11 boys from 9 families with an increased urinary Cr:Crn ratio (18%). In three related boys, a hemizygous missense mutation (c.1271G>A; p.Gly424Asp) was identified. Their mother was heterozygous for the same mutation. Although many missense mutations have been described, the p.Gly424Asp mutation has not been previously reported. The clinical expression varied widely among affected males of this family. Patients 1 and 3 had relatively mild clinical expression (mild mental retardation (MR) and attention deficit disorder), but patient 2 had all typical clinical signs of SLC6A8 defect such as moderate MR, autistic features, expressive dysphasia and epilepsy. Among our patients, we saw significant problems in speech and language development combined with attention and behavioural difficulties. The number of false-positive biochemical results with increased urinary Cr:Crn ratio was higher (18%) in our study than in previous reports (1.8­10%). We therefore suggest that repeated biochemical testing should be performed before DNA sequencing analysis. Our study suggests that 2% (95% confidence limits: 0.05­11.1%) of this Estonian XLMR panel are due to mutations in the SLC6A8, which is similar to the prevalence reported in other populations. We therefore conclude that creatine transporter deficiency is a relatively common genetic disorder in males with sporadic or familiar MR and diagnostic screening of them should always include screening for SLC6A8 deficiency.