Dodecyl creatine ester therapy: from promise to reality.

Pathogenic variants in SLC6A8, the gene which encodes creatine transporter SLC6A8, prevent creatine uptake in the brain and result in a variable degree of intellectual disability, behavioral disorders (e.g., autism spectrum disorder), epilepsy, and severe speech and language delay. There are no treatments to improve neurodevelopmental outcomes for creatine transporter deficiency (CTD). In this spotlight, we summarize recent advances in innovative molecules to treat CTD, with a focus on dodecyl creatine ester, the most promising drug candidate.

Triac Treatment Prevents Neurodevelopmental and Locomotor Impairments in Thyroid Hormone Transporter Mct8/Oatp1c1 Deficient Mice.

Patients with inactive thyroid hormone (TH) transporter MCT8 display intellectual disability due to compromised central TH transport and action. As a therapeutic strategy, application of thyromimetic, MCT8-independent compounds Triac (3,5,3'-triiodothyroacetic acid), and Ditpa (3,5-diiodo-thyropropionic acid) was proposed. Here, we directly compared their thyromimetic potential in Mct8/Oatp1c1 double knock-out mice (Dko) modeling human MCT8 deficiency. Dko mice received either Triac (50 ng/g or 400 ng/g) or Ditpa (400 ng/g or 4000 ng/g) daily during the first three postnatal weeks. Saline-injected Wt and Dko mice served as controls. A second cohort of Dko mice received Triac (400 ng/g) daily between postnatal weeks 3 and 6. Thyromimetic effects were assessed at different postnatal stages by immunofluorescence, ISH, qPCR, electrophysiological recordings, and behavior tests. Triac treatment (400 ng/g) induced normalized myelination, cortical GABAergic interneuron differentiation, electrophysiological parameters, and locomotor performance only when administered during the first three postnatal weeks. Ditpa (4000 ng/g) application to Dko mice during the first three postnatal weeks resulted in normal myelination and cerebellar development but only mildly improved neuronal parameters and locomotor function. Together, Triac is highly-effective and more efficient than Ditpa in promoting CNS maturation and function in Dko mice yet needs to be initiated directly after birth for the most beneficial effects.

TRIAC Treatment Improves Impaired Brain Network Function and White Matter Loss in Thyroid Hormone Transporter Mct8/Oatp1c1 Deficient Mice.

Dysfunctions of the thyroid hormone (TH) transporting monocarboxylate transporter MCT8 lead to a complex X-linked syndrome with abnormal serum TH concentrations and prominent neuropsychiatric symptoms (Allan-Herndon-Dudley syndrome, AHDS). The key features of AHDS are replicated in double knockout mice lacking MCT8 and organic anion transporting protein OATP1C1 (Mct8/Oatp1c1 DKO). In this study, we characterize impairments of brain structure and function in Mct8/Oatp1c1 DKO mice using multimodal magnetic resonance imaging (MRI) and assess the potential of the TH analogue 3,3',5-triiodothyroacetic acid (TRIAC) to rescue this phenotype. Structural and functional MRI were performed in 11-weeks-old male Mct8/Oatp1c1 DKO mice (N = 10), wild type controls (N = 7) and Mct8/Oatp1c1 DKO mice (N = 13) that were injected with TRIAC (400 ng/g bw s.c.) daily during the first three postnatal weeks. Grey and white matter volume were broadly reduced in Mct8/Oatp1c1 DKO mice. TRIAC treatment could significantly improve white matter thinning but did not affect grey matter loss. Network-based statistic showed a wide-spread increase of functional connectivity, while graph analysis revealed an impairment of small-worldness and whole-brain segregation in Mct8/Oatp1c1 DKO mice. Both functional deficits could be substantially ameliorated by TRIAC treatment. Our study demonstrates prominent structural and functional brain alterations in Mct8/Oatp1c1 DKO mice that may underlie the psychomotor deficiencies in AHDS. Additionally, we provide preclinical evidence that early-life TRIAC treatment improves white matter loss and brain network dysfunctions associated with TH transporter deficiency.

Current and potential new treatment strategies for creatine deficiency syndromes.

Creatine deficiency syndromes (CDS) are inherited metabolic disorders caused by mutations in GATM, GAMT and SLC6A8 and mainly affect central nervous system (CNS). AGAT- and GAMT-deficient patients lack the functional brain endogenous creatine (Cr) synthesis pathway but express the Cr transporter SLC6A8 at blood-brain barrier (BBB), and can thus be treated by oral supplementation of high doses of Cr. For Cr transporter deficiency (SLC6A8 deficiency or CTD), current treatment strategies benefit one-third of patients. However, as their phenotype is not completely reversed, and for the other two-thirds of CTD patients, the development of novel more effective therapies is needed. This article aims to review the current knowledge on Cr metabolism and CDS clinical aspects, highlighting their current treatment possibilities and the most recent research perspectives on CDS potential therapeutics designed, in particular, to bring new options for the treatment of CTD.

Long-Term Efficacy of T3 Analogue Triac in Children and Adults With MCT8 Deficiency: A Real-Life Retrospective Cohort Study.

CONTEXT: Patients with mutations in thyroid hormone transporter MCT8 have developmental delay and chronic thyrotoxicosis associated with being underweight and having cardiovascular dysfunction. OBJECTIVE: Our previous trial showed improvement of key clinical and biochemical features during 1-year treatment with the T3 analogue Triac, but long-term follow-up data are needed. METHODS: In this real-life retrospective cohort study, we investigated the efficacy of Triac in MCT8-deficient patients in 33 sites. The primary endpoint was change in serum T3 concentrations from baseline to last available measurement. Secondary endpoints were changes in other thyroid parameters, anthropometric parameters, heart rate, and biochemical markers of thyroid hormone action. RESULTS: From October 15, 2014 to January 1, 2021, 67 patients (median baseline age 4.6 years; range, 0.5-66) were treated up to 6 years (median 2.2 years; range, 0.2-6.2). Mean T3 concentrations decreased from 4.58 (SD 1.11) to 1.66 (0.69) nmol/L (mean decrease 2.92 nmol/L; 95% CI, 2.61-3.23; P < 0.0001; target 1.4-2.5 nmol/L). Body-weight-for-age exceeded that of untreated historical controls (mean difference 0.72 SD; 95% CI, 0.36-1.09; P = 0.0002). Heart-rate-for-age decreased (mean difference 0.64 SD; 95% CI, 0.29-0.98; P = 0.0005). SHBG concentrations decreased from 245 (99) to 209 (92) nmol/L (mean decrease 36 nmol/L; 95% CI, 16-57; P = 0.0008). Mean creatinine concentrations increased from 32 (11) to 39 (13) µmol/L (mean increase 7 µmol/L; 95% CI, 6-9; P < 0.0001). Mean creatine kinase concentrations did not significantly change. No drug-related severe adverse events were reported. CONCLUSIONS: Key features were sustainably alleviated in patients with MCT8 deficiency across all ages, highlighting the real-life potential of Triac for MCT8 deficiency.

Development of a Redox-Sensitive Spermine Prodrug for the Potential Treatment of Snyder Robinson Syndrome.

Snyder Robinson Syndrome (SRS) is a rare disease associated with a defective spermine synthase gene and low intracellular spermine levels. In this study, a spermine replacement therapy was developed using a spermine prodrug that enters cells via the polyamine transport system. The prodrug was comprised of three components: a redox-sensitive quinone "trigger", a "trimethyl lock (TML)" aryl "release mechanism", and spermine. The presence of spermine in the design facilitated uptake by the polyamine transport system. The quinone-TML motifs provided a redox-sensitive agent, which upon intracellular reduction generated a hydroquinone, which underwent intramolecular cyclization to release free spermine and a lactone byproduct. Rewardingly, most SRS fibroblasts treated with the prodrug revealed a significant increase in intracellular spermine. Administering the spermine prodrug through feeding in a Drosophila model of SRS showed significant beneficial effects. In summary, a spermine prodrug is developed and provides a lead compound for future spermine replacement therapy experiments.

CNKSR2 gene mutation leads to Houge type of X-linked syndromic mental retardation: A case report and review of literature.

RATIONALE: Mutations of connector enhancer of kinase suppressor of Ras-2 (CNKSR2) gene were identified as the cause of Houge type of X-linked syndromic mental retardation. The mutations of CNKSR2 gene are rare, we reporta patient carrying a novel nonsense mutation of CNKSR2,c.625C > T(p.Gln209∗) and review the clinical features and mutations of CNKSR2 gene for this rare condition considering previous literature. PATIENT CONCERNS: We report a case of a 7-year and 5-month-old Chinese patient with clinical symptoms of intellectual disability, language defect, epilepsy and hyperactivity. Genetic study revealed a novel nonsense variant of CNKSR2, which has not been reported yet. DIAGNOSIS: According to clinical manifestations, genetic pattern and ACMG classification of mutation site as Class 1-cause disease, the patient was diagnosed as Houge type of X-linked syndromic mental retardation caused by CNKSR2 gene mutation. INTERVENTIONS: The patient was administrated with a gradual titration of valproic acid (VPA). OUTCOMES: On administration of valproic acid, he had no further seizures. LESSONS: This is the first time to report a nonsense variant in CNKSR2, c.625C > T(p.Gln209∗), this finding could expand the spectrum of CNKSR2 mutations and might also support the further study of Houge type of X-linked syndromic mental retardation.

Identification of Inhibitors Based on Molecular Docking: Thyroid Hormone Transmembrane Transporter MCT8 as a Target.

AIMS: To identify natural inhibitors against MCT8 for Allan-Herndon-Dudley Syndrome. BACKGROUND: Monocarboxylate Transporter 8 (MCT8) is a Thyroid Hormone (TH) transporter which is highly expressed in the liver and brain. Mutations in the MCT8 gene (SLC16A2) cause a syndrome of psychomotor retardation in humans, known as Allan-Herndon-Dudley syndrome (AHDS). Currently, no treatment is available for AHDS. Therefore, there is a need to discover new inhibitors of MCT8 for treating AHDS. OBJECTIVE: Considering the importance of natural compounds in drug discovery, this study aimed to identify potential natural inhibitors against MCT8. METHODS: As Protein-ligand interactions play a key role in structure based drug design, this study screened 24 natural kinase inhibitors and investigated their binding affinity against MCT8 by using molecular docking. The modelled 3D structure of MCT8 docked with 24 compounds using PyRX through Autodock Vina. Drug-likeness studies were made using Swiss ADME and Lipinski's rule of five was performed. Triac, desipramine and silychristin were used as the positive controls. Binding energies of the selected compounds were compared with that of positive controls. RESULT: The results showed that emodin exhibited best binding energy of -8.6 kcal/mol followed by helenaquinol, cercosporamide and resveratrol. Moreover, it was observed that emodin and helenaquinol exhibit higher binding energy than the positive controls. Cercosporamide and resveratrol exhibited higher binding energy than triac and desipramine and showed the binding energy similar to silychristin. CONCLUSION: This study reveals that these compounds could be promising candidates for further evaluation for AHDS prevention.

Prenatal Treatment of Thyroid Hormone Cell Membrane Transport Defect Caused by MCT8 Gene Mutation.

Background: Mutations of the thyroid hormone (TH)-specific cell membrane transporter, monocarboxylate transporter 8 (MCT8), produce an X-chromosome-linked syndrome of TH deficiency in the brain and excess in peripheral tissues. The clinical consequences include brain hypothyroidism causing severe psychoneuromotor abnormalities (no speech, truncal hypotonia, and spastic quadriplegia) and hypermetabolism (poor weight gain, tachycardia, and increased metabolism, associated with high serum levels of the active TH, T3). Treatment in infancy and childhood with TH analogues that reduce serum triiodothyronine (T3) corrects hypermetabolism, but has no effect on the psychoneuromotor deficits. Studies of brain from a 30-week-old MCT8-deficient embryo indicated that brain abnormalities were already present during fetal life. Methods: A carrier woman with an affected male child (MCT8 A252fs268*), pregnant with a second affected male embryo, elected to carry the pregnancy to term. We treated the fetus with weekly 500 µg intra-amniotic instillation of levothyroxine (LT4) from 18 weeks of gestation until birth at 35 weeks. Thyroxine (T4), T3, and thyrotropin (TSH) were measured in the amniotic fluid and maternal serum. Treatment after birth was continued with LT4 and propylthiouracil. Follow-up included brain magnetic resonance imaging (MRI) and neurodevelopmental evaluation, both compared with the untreated brother. Results: During intrauterine life, T4 and T3 in the amniotic fluid were maintained above threefold to twofold the baseline and TSH was suppressed by 80%, while maternal serum levels remained unchanged. At birth, the infant serum T4 was 14.5 µg/dL and TSH <0.01 mU/L compared with the average in untreated MCT8-deficient infants of 5.1 µg/ and >8 mU/L, respectively. MRI at six months of age showed near-normal brain myelination compared with much reduced in the untreated brother. Neurodevelopmental assessment showed developmental quotients in receptive language and problem-solving, and gross motor and fine motor function ranged from 12 to 25 at 31 months in the treated boy and from 1 to 7 at 58 months in the untreated brother. Conclusions: This is the first demonstration that prenatal treatment improved the neuromotor and neurocognitive function in MCT8 deficiency. Earlier treatment with TH analogues that concentrate in the fetus when given to the mother may further rescue the phenotype.

Creatine Transporter Deficiency Presenting as Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is the most common disability-causing neurodevelopmental disorder in childhood. Although inborn errors of metabolism (IEM) are rare causes of ASD, they are significant for several reasons, including implications in genetic counseling and determination of prognosis. In this article, we present a 6-year-old boy who presented to us with ASD and was diagnosed with creatine transporter deficiency. Physical and neurologic examination of this patient had not previously raised suspicion of IEM, but twin pregnancy, prematurity, NICU stay due to necrotizing enterocolitis, transient infantile hypotonia, gross-motor delay, breath-holding spells, and a single febrile seizure complicated the history. MRI revealed mild T2-hyperintensity in posterior periventricular white matter. Further evaluation with magnetic resonance spectroscopy, which showed a decreased creatine peak, led to diagnostic investigations for disorders of creatine metabolism, revealing increased urinary creatine:creatinine ratio and a de novo, novel hemizygous frameshift variant in SLC6A8 Clinicians are advised to maintain a high index of suspicion for IEM and to evaluate patients with ASD for syndromic features. Although current guidelines from relevant organizations differ in their recommendations regarding the necessity and the extent of metabolic screening in ASD, there is a growing trend toward screening for treatable IEM. In this case report, we present challenges and pitfalls in the diagnostic journey for creatine transporter deficiency and underline the significance of a thorough history and physical examination in the evaluation of a child with ASD.

Cyclocreatine treatment ameliorates the cognitive, autistic and epileptic phenotype in a mouse model of Creatine Transporter Deficiency.

Creatine Transporter Deficiency (CTD) is an inborn error of metabolism presenting with intellectual disability, behavioral disturbances and epilepsy. There is currently no cure for this disorder. Here, we employed novel biomarkers for monitoring brain function, together with well-established behavioral readouts for CTD mice, to longitudinally study the therapeutic efficacy of cyclocreatine (cCr) at the preclinical level. Our results show that cCr treatment is able to partially correct hemodynamic responses and EEG abnormalities, improve cognitive deficits, revert autistic-like behaviors and protect against seizures. This study provides encouraging data to support the potential therapeutic benefit of cyclocreatine or other chemically modified lipophilic analogs of Cr.

Immunologic phenotype of a child with the MEHMO syndrome.

MEHMO syndrome is a rare X-linked syndrome characterized by Mental retardation, Epilepsy, Hypogenitalism, Microcephaly, and Obesity associated with the defect of protein synthesis caused by the EIF2S3 gene mutations. We hypothesized that the defect in protein synthesis could have an impact on the immune system. We describe immunologic phenotype and possible treatment outcomes in patient with MEHMO syndrome carrying a frame-shift mutation (I465fs) in the EIF2S3 gene. The proband (currently 9-year-old boy) had normal IgG and IgM levels, but had frequent respiratory and urinary tract infections. On subcutaneous immunoglobulin therapy achieving supra-physiological IgG levels the frequency of infections significantly decreased in Poisson regression by 54.5 % (CI 33.2-89.7, p=0.017). The MEHMO patient had had frequent acute infections despite normal IgG and IgM serum levels and responded well to the immunoglobulin treatment.

Allan-Herndon-Dudley-Syndrome: Considerations about the Brain Phenotype with Implications for Treatment Strategies.

Despite its first description more than 75 years ago, effective treatment for "Allan-Herndon-Dudley-Syndrome (AHDS)", an X-linked thyroid hormone transporter defect, is unavailable. Mutations in the SLC16A2 gene have been discovered to be causative for AHDS in 2004, but a comprehensive understanding of the function of the encoded protein, monocarboxylate transporter 8 (MCT8), is incomplete. Patients with AHDS suffer from neurodevelopmental delay, as well as extrapyramidal (dystonia, chorea, athetosis), pyramidal (spasticity), and cerebellar symptoms (ataxia). This suggests an affection of the pyramidal tracts, basal ganglia, and cerebellum, most likely already during fetal brain development. The function of other brain areas relevant for mood, behavior, and vigilance seems to be intact. An optimal treatment strategy should thus aim to deliver T3 to these relevant structures at the correct time points during development. A potential therapeutic strategy meeting these needs might be the delivery of T3 via a "Trojan horse mechanism" by which T3 is delivered into target cells by a thyroid hormone transporter independent T3 internalization.

Pharmacological prospects of G-quadruplexes for neurological diseases using porphyrins.

Genomic regions with guanine (G)-rich sequences make non-Watson-Crick base pairs, which result in the formation of unique nucleic acid structures called G-quadruplexes (G4s) in cells. Studies have suggested that abnormal G4s are involved in neurological diseases. For example, the formation of G4s caused by expansion of G-rich sequences is implicated in C9orf72-mediated amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), and fragile X-related tremor/ataxia syndrome (FXTAS). In addition, the disruption and/or mutation of G4 binding proteins (G4BPs), such as heterogeneous nuclear ribonucleoproteins (hnRNPs) and DNA/RNA helicases, is related to neurological diseases. For instance, mutations in a G4BP called ATRX lead to a neurodevelopmental disorder, ATR-X syndrome, which is associated with intellectual disability. We found that porphyrins are potential candidate drugs for treating ATR-X syndrome through their G4 binding ability. Importantly, intracellular porphyrins are produced from 5-aminolevulinic acid (5-ALA) in vivo. Oral administration of 5-ALA improved cognitive dysfunction in an ATR-X syndrome model mouse, and language ability in an ATR-X syndrome patient. In this review, we suggest a novel therapeutic strategy targeting G4s using porphyrins in neurological diseases.

Phosphocyclocreatine is the dominant form of cyclocreatine in control and creatine transporter deficiency patient fibroblasts.

Creatine transporter deficiency (CTD) is a metabolic disorder resulting in cognitive, motor, and behavioral deficits. Cyclocreatine (cCr), a creatine analog, has been explored as a therapeutic strategy for the treatment of CTD. We developed a rapid, selective, and accurate HILIC ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify the intracellular concentrations of cCr, creatine (Cr), creatine-d3 (Cr-d3), phosphocyclocreatine (pcCr), and phosphocreatine (pCr). Using HILIC-UPLC-MS/MS, we measured cCr and Cr-d3 uptake and their conversion to the phosphorylated forms in primary human control and CTD fibroblasts. Altogether, the data demonstrate that cCr enters cells and its dominant intracellular form is pcCr in both control and CTD patient cells. Therefore, cCr may replace creatine as a therapeutic strategy for the treatment of CTD.

Effectiveness and safety of the tri-iodothyronine analogue Triac in children and adults with MCT8 deficiency: an international, single-arm, open-label, phase 2 trial.

BACKGROUND: Deficiency of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) causes severe intellectual and motor disability and high serum tri-iodothyronine (T3) concentrations (Allan-Herndon-Dudley syndrome). This chronic thyrotoxicosis leads to progressive deterioration in bodyweight, tachycardia, and muscle wasting, predisposing affected individuals to substantial morbidity and mortality. Treatment that safely alleviates peripheral thyrotoxicosis and reverses cerebral hypothyroidism is not yet available. We aimed to investigate the effects of treatment with the T3 analogue Triac (3,3',5-tri-iodothyroacetic acid, or tiratricol), in patients with MCT8 deficiency. METHODS: In this investigator-initiated, multicentre, open-label, single-arm, phase 2, pragmatic trial, we investigated the effectiveness and safety of oral Triac in male paediatric and adult patients with MCT8 deficiency in eight countries in Europe and one site in South Africa. Triac was administered in a predefined escalating dose schedule-after the initial dose of once-daily 350 µg Triac, the daily dose was increased progressively in 350 µg increments, with the goal of attaining serum total T3 concentrations within the target range of 1·4-2·5 nmol/L. We assessed changes in several clinical and biochemical signs of hyperthyroidism between baseline and 12 months of treatment. The prespecified primary endpoint was the change in serum T3 concentrations from baseline to month 12. The co-primary endpoints were changes in concentrations of serum thyroid-stimulating hormone (TSH), free and total thyroxine (T4), and total reverse T3 from baseline to month 12. These analyses were done in patients who received at least one dose of Triac and had at least one post-baseline evaluation of serum throid function. This trial is registered with ClinicalTrials.gov, number NCT02060474. FINDINGS: Between Oct 15, 2014, and June 1, 2017, we screened 50 patients, all of whom were eligible. Of these patients, four (8%) patients decided not to participate because of travel commitments. 46 (92%) patients were therefore enrolled in the trial to receive Triac (median age 7·1 years [range 0·8-66·8]). 45 (98%) participants received Triac and had at least one follow-up measurement of thyroid function and thus were included in the analyses of the primary endpoints. Of these 45 patients, five did not complete the trial (two patients withdrew [travel burden, severe pre-existing comorbidity], one was lost to follow-up, one developed of Graves disease, and one died of sepsis). Patients required a mean dose of 38.3 µg/kg of bodyweight (range 6·4-84·3) to attain T3 concentrations within the target range. Serum T3 concentration decreased from 4·97 nmol/L (SD 1·55) at baseline to 1·82 nmol/L (0·69) at month 12 (mean decrease 3·15 nmol/L, 95% CI 2·68-3·62; p<0·0001), while serum TSH concentrations decreased from 2·91 mU/L (SD 1·68) to 1·02 mU/L (1·14; mean decrease 1·89 mU/L, 1·39-2·39; p<0·0001) and serum free T4 concentrations decreased from 9·5 pmol/L (SD 2·5) to 3·4 (1·6; mean decrease 6·1 pmol/L (5·4-6·8; p<0·0001). Additionally, serum total T4 concentrations decreased by 31·6 nmol/L (28·0-35·2; p<0·0001) and reverse T3 by 0·08 nmol/L (0·05-0·10; p<0·0001). Seven treatment-related adverse events (transiently increased perspiration or irritability) occurred in six (13%) patients. 26 serious adverse events that were considered unrelated to treatment occurred in 18 (39%) patients (mostly hospital admissions because of infections). One patient died from pulmonary sepsis leading to multi-organ failure, which was unrelated to Triac treatment. INTERPRETATION: Key features of peripheral thyrotoxicosis were alleviated in paediatric and adult patients with MCT8 deficiency who were treated with Triac. Triac seems a reasonable treatment strategy to ameliorate the consequences of untreated peripheral thyrotoxicosis in patients with MCT8 deficiency. FUNDING: Dutch Scientific Organization, Sherman Foundation, NeMO Foundation, Wellcome Trust, UK National Institute for Health Research Cambridge Biomedical Centre, Toulouse University Hospital, and Una Vita Rara ONLUS.

Dodecyl creatine ester-loaded nanoemulsion as a promising therapy for creatine transporter deficiency.

Creatine transporter (CrT) deficiency is an X-linked intellectual disability caused by mutations of CrT. Aim: This work focus on the preclinical development of a new therapeutic approach based on a microemulsion (ME) as drug delivery system for dodecyl creatine ester (DCE). Materials & methods: DCE-ME was prepared by titration method. Novel object recognition (NOR) tests were performed before and after DCE-ME treatment on Slc6a8-/y mice. Results: Intranasal administration with DCE-ME improved NOR performance in Slc6a8-/y mice. Slc6a8-/y mice treated with DCE-ME had increased striatal ATP levels mainly in the striatum compared with vehicle-treated Slc6a8-/y mice which was associated with increased expression of synaptic markers. Conclusion: These results highlight the potential value of DCE-ME as promising therapy for creatine transporter deficiency.

Rescue by 4-phenylbutyrate of several misfolded creatine transporter-1 variants linked to the creatine transporter deficiency syndrome.

Diseases arising from misfolding of SLC6 transporters have been reported over recent years, e.g. folding-deficient mutants of the dopamine transporter and of the glycine transporter-2 cause infantile/juvenile Parkinsonism dystonia and hyperekplexia, respectively. Mutations in the coding sequence of the human creatine transporter-1 (hCRT-1/SLC6A8) gene result in a creatine transporter deficiency syndrome, which varies in its clinical manifestation from epilepsy, mental retardation, autism, development delay and motor dysfunction to gastrointestinal symptoms. Some of the mutations in hCRT-1 occur at residues, which are highly conserved across the SLC6 family. Here, we examined 16 clinically relevant hCRT-1 variants to verify the conjecture that they were misfolded and that this folding defect was amenable to correction. Confocal microscopy imaging revealed that the heterologously expressed YFP-tagged mutant CRTs were trapped in the endoplasmic reticulum (ER), co-localised with the ER-resident chaperone calnexin. In contrast, the wild type hCRT-1 reached the plasma membrane. Preincubation of transiently transfected HEK293 cells with the chemical chaperone 4-phenylbutyrate (4-PBA) restored ER export and surface expression of as well as substrate uptake by several folding-deficient CRT-1 mutants. A representative mutant (hCRT-1-P544L) was expressed in rat primary hippocampal neurons to verify pharmacochaperoning in a target cell: 4-PBA promoted the delivery of hCRT-1-P544L to the neurite extensions. These observations show that several folding-deficient hCRT-1 mutants can be rescued. This proof-of-principle justifies the search for additional pharmacochaperones to restore folding of 4PBA-unresponsive hCRT-1 mutants. Finally, 4-PBA is an approved drug in paediatric use: this provides a rationale for translating the current insights into clinical trials. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

A Novel Nonsense Mutation of PHF6 in a Female with Extended Phenotypes of Borjeson-Forssman-Lehmann Syndrome

Borjeson-Forssman-Lehmann syndrome (BFLS) is a rare X-linked disease caused by PHF6 mutations. Classic BFLS has been associated with intellectual disability (ID), developmental delay (DD), obesity, epilepsy, typical facial features and anomalies of fingers and toes. Endocrinological phenotypes and outcome of treatment in this condition remain to be delineated. Here we report a patient who exhibited complete growth hormone deficiency who responded to hormonal treatment but with adverse effects. Horseshoe kidney was present in this patient, which is also atypical in BFLS. A heterozygous nonsense mutation c.673C>T (p.R225X) of PHF6 gene was identified in the patient, inherited from her unaffected mother. Both the patient and her mother showed highly skewed X-inactivation. We reviewed the phenotypes of all reported BFLS cases, and summarized their endocrine presentations. This first report of an Asian patient with BFLS further delineated the genetic and phenotypic spectrum of the syndrome. The adverse effect experienced by the patient suggests caution in the use of growth hormone treatment in this condition.