X-linked creatine-transporter gene (SLC6A8) defect: a new creatine-deficiency syndrome

We report the first X-linked creatine-deficiency syndrome caused by a defective creatine transporter. The male index patient presented with developmental delay and hypotonia. Proton magnetic-resonance spectroscopy of his brain revealed absence of the creatine signal. However, creatine in urine and plasma was increased, and guanidinoacetate levels were normal. In three female relatives of the index patient, mild biochemical abnormalities and learning disabilities were present, to various extents. Fibroblasts from the index patient contained a hemizygous nonsense mutation in the gene SLC6A8 and were defective in creatine uptake. The three female relatives were heterozygous for this mutation in SLC6A8, which has been mapped to Xq28.     

Presence of normal creatine in the muscle of a patient with a mutation in the creatine transporter: A case study

To date, more than seven families have been reported who carry a mutation in the X-linked creatine-transporter (CrT) gene. The resulting lack of creatine in the brain is associated with mental retardation, severe expressive language disorder, mild epilepsy, and a complete absence of Cr in the brain (measured using MRS). Conversely, these patients had no observable cardiac or musculo-skeletal deficits. In this case study, a 22-year-old patient underwent surgical repair for scoliosis. Proton MRS of this patient's brain demonstrated the near-absence of creatine and phosphocreatine within the cerebral white and deep gray matter structures. Cerebral atrophy was noted with serial MRI examinations. Subsequent genetic and metabolic analysis showed some biochemical anomalies consistent with a CrT deficiency. The mutation in this patient was identified as a deletion at phenylalanine 107 (delF107). Control muscle biopsies were obtained from archived samples, which had been taken with informed consent during routine muscle biopsies for diagnostic purposes. We determined that the total Cr concentration in the skeletal muscle biopsy was 39.3 +/- 2.94 mmol/kg wet wt., which is not significantly different from non-CrT controls, n = 3 (43.3 +/- 3.57 mmol/kg wet wt.). We conclude that the brain appears to lack the ability to transport creatine when there is a mutation in the CrT gene. However, the muscle utilizes another mechanism for maintaining normal creatine levels. Identifying this alternative creatine-transport mechanism may be useful in treating the neurologic and cognitive impairments of patients with creatine-transporter deficiency.     

Neuropsychological profile and clinical effects of arginine treatment in children with creatine transport deficiency

ABSTRACT: BACKGROUND: SLC6A8, an X-linked gene, encodes the creatine transporter (CRTR) and its mutations lead to cerebral creatine (Cr) deficiency which results in mental retardation, speech and language delay, autistic-like behaviour and epilepsy (CRTR-D, OMIM 300352). CRTR-D represents the most frequent Cr metabolism disorder but, differently from Cr synthesis defects, that are partially reversible by oral Cr supplementation, does not respond to Cr treatment even if precociously administrated. The precursors of Cr are the non-essential amino acids Glycine (Gly) and Arginine (Arg), which have their own transporters at the brain-blood barrier level and, therefore, their supplementation appears an attractive and feasible therapeutic option aimed at stimulating Cr endogenous synthesis and, in this way, at overcoming the block of Cr transport within the brain. However, until now the effects of Arg and/or Gly supplementation on Cr brain levels and behaviour have been controversial. METHODS: In this study five Italian male patients affected by CRTR-D were supplemented with oral LArg at a dosage of 300 mg/kg/day divided into 3 doses, for 24-36 months. Biochemical and plasmatic amino acids examinations and thyroid hormone dosages were periodically performed. Moreover, Proton and Phosphorus Magnetic Resonance Spectroscopy (MRS) was monitored during follow-up in concurrence with neuropsychological evaluations. RESULTS: During L-Arg treatment a clinical improvement in motor skills and to a lesser extent in communication and attention was observed. In addition, all patients had a reduction in the number and frequency of epileptic seizures. Daily living skills appeared also to be positively influenced by L-Arg treatment. Moreover, Total Cr and especially PhosphoCr, evaluated byproton and phosphorus spectroscopy, showed a mild increase, although well below the normal range. CONCLUSION: This study provides information to support the effectiveness of L-Arg supplement treatment in CTRT-D patients; in fact the syndromic pattern of cognitive and linguistic deficit presented by CRTR-D patients was partially altered by L-Arg supplementation especially at a qualitative clinical level. Oral L-Arg may represent not only a protective factor towards a further cognitive decline, but can lead to the acquisition of new skills.     

肌肉31P-MRS 的临床研究进展

磁共振波谱分析(magnetic resonance spectroscopy MRS)是目前唯一无创性定量研究人体组织细胞代谢、生理生化改变的方法。磁共振磷谱(31P-MRS)可对无机磷(Pi)、磷酸肌酸(PCr)、三磷酸腺苷(ATP)等含磷高能化合物进行定量分析,是在体研究骨骼肌能量代谢的有力工具。动态磷谱技术可测量肌肉在静息状态、收缩过程和恢复过程中细胞内高能磷酸化合物的变化,评价骨骼肌做功时的能量的转换效率,实现对线粒体功能的无创性评价。本文将对肌肉磷谱的研究进展做综述,尤其侧重于动态磷谱的应用,为以后利用磷谱客观研究肌肉相关疾病奠定良好的基础。     

A de novo 2.3 Mb deletion in 2q24.2q24.3 in a 20-month-old developmentally delayed girl

We report a 20-month-old girl ascertained at the age of 11 months for developmental delay. She presented with hypotonia and delayed motor development. The patient had severe language impairment and showed behaviour consistent with autism spectrum disorder. She was microcephalic with mild dysmorphic features and had joint hyperlaxity. We detected a 2.3 Mb de novo deletion in 2q24.2q24.3 on her paternal chromosome.     

Effects of Amide Creatine Derivatives in Brain Hippocampal Slices, and Their Possible Usefulness for Curing Creatine Transporter Deficiency

The creatine/phosphocreatine system carries ATP from production to consumption sites and buffers the intracellular content of ATP at times of energy deprivation. The creatine transporter deficiency syndrome is an X-linked disease caused by a defective creatine transporter into the central nervous system. This disease is presently untreatable because creatine lacking its carrier cannot cross neither the blood–brain barrier nor the cell plasma membranes. Possible strategies to cure this condition are to couple creatine to molecules which have their own carrier, to exploit the latter to cross biological membranes or to modify the creatine molecule to make it more lipophilic, in such a way that it may more easily cross lipid-rich biological membranes. Such molecules could moreover be useful for treatment of stroke or other ischemic brain syndromes of normal (transporter working) tissue. In this paper we tested four molecules in in vitro hippocampal slices experiments to investigate whether or not they had a neuroprotective effect similar to that of creatine. On two of them we also performed biochemical measurements to investigate whether or not they were able to increase the creatine and phosphocreatine content of the hippocampal slices with and without block of the transporter. We found that these molecules increase levels of creatine after block of the transporter, and significantly increased the levels of phosphocreatine. Both significantly increased the total creatine content in both conditions of active and blocked transporter. This shows that these molecules are capable of entering cells through biological membranes without using the creatine transporter. By contrast, neither of them was able to delay synaptic block during anoxia of normal (transporter functioning) tissue. We conclude that these compounds might possibly be useful for therapy of creatine transporter deficiency, but further research is needed to understand their possible role in anoxia/ischemia of normal tissue.     

Neurodevelopmental outcome in children with periventricular leukomalacia

The purpose of this study was to question the correlation of different grades of periventricular leukomalacia (PVL) and subsequent neurodevelopmental outcome. In a prospective study we followed 52 preterm infants. Infants were divided into three groups according to their cranial ultrasound findings of PVL (De Vries classification). Seventeen children had PVL 1, 20 children had PVL 2, and 15 children had PVL 3. All 15 (100%) children with PVL 3 developed cerebral palsy with additional visual perceptual dysfunctions and epilepsy. Children with PVL 1 had high frequency of mild neuromotoric delay and visual impairment. PVL 2 and 3 have great predictive value for subsequent severe neurodevelopmental disorder which refers to cerebral palsy, different cognitive deficits, vision impairment and epilepsy. We have determined that due to high frequency of visual impairment and epilepsy we need to include neurophysiologic examinations very early in children with PVL lesions.     

肌肉~（31）P-MRS的临床研究进展

磁共振波谱分析（magnetic resonance spectroscopy MRS）是目前唯一无创性定量研究人体组织细胞代谢、生理生化改变的方法。磁共振磷谱（31P-MRS）可对无机磷（Pi）、磷酸肌酸（PCr）、三磷酸腺苷（ATP）等含磷高能化合物进行定量分析,是在体研究骨骼肌能量代谢的有力工具。动态磷谱技术可测量肌肉在静息状态、收缩过程和恢复过程中细胞内高能磷酸化合物的变化,评价骨骼肌做功时的能量的转换效率,实现对线粒体功能的无创性评价。本文将对肌肉磷谱的研究进展做综述,尤其侧重于动态磷谱的应用,为以后利用磷谱客观研究肌肉相关疾病奠定良好的基础。     

The role of pH on the thermodynamics and kinetics of muscle biochemistry: an in vivo study by (31)P-MRS in patients with myo-phosphorylase deficiency

In this study we assessed ΔG'(ATP) hydrolysis, cytosolic [ADP], and the rate of phosphocreatine recovery using Phosphorus Magnetic Resonance Spectroscopy in the calf muscle of a group of patients affected by glycogen myo-phosphorylase deficiency (McArdle disease). The goal was to ascertain whether and to what extent the deficit of the glycogenolytic pathway would affect the muscle energy balance. A typical feature of this pathology is the lack of intracellular acidosis. Therefore we posed the question of whether, in the absence of pH decrease, the rate of phosphocreatine recovery depends on the amount of phosphocreatine consumed during exercise. Results showed that at the end of exercise both [ADP] and ΔG'(ATP) of patients were significantly higher than those of matched control groups reaching comparable levels of phosphocreatine concentration. Furthermore, in these patients we found that the rate of phosphocreatine recovery is not influenced by the amount of phosphocreatine consumed during exercise. These outcomes provide experimental evidence that: i) the intracellular acidification occurring in exercising skeletal muscle is a protective factor for the energy consumption; and ii) the influence of pH on the phosphocreatine recovery rate is at least in part related to the kinetic mechanisms of mitochondrial creatine kinase enzyme.     

Arginine and glycine stimulate creatine synthesis in creatine transporter 1-deficient lymphoblasts

Creatine transporter 1 (CT1) defect is an X-linked disease that causes severe neurological impairment. No treatment has been available for this condition so far. Because the transport of creatine (Cr) precursors Gly and Arg is not affected in this disorder, we tested the possible corrective effect of these two amino acids on Cr depletion in lymphoblasts lacking the transporter. Substrates enriched with Arg or Arg plus Gly increased the concentration of intracellular Cr in affected cells as well as in control cells. The greatest effect was obtained with 10 and 15 mM Arg and 10mM Arg plus Gly. These results encourage an in vivo trial with Cr precursors in CT1 defect.     

Mowat-Wilson syndrome in a Moroccan consanguineous family

Mowat-Wilson syndrome is a mental retardation-multiple congenital anomaly syndrome characterized by a typical facies, developmental delay, epilepsy, and variable congenital malformations, including Hirschsprung disease, urogenital anomalies, congenital heart disease, and agenesis of the corpus callosum. This disorder is sporadic and is caused by heterozygous mutations or deletions of the ZFHX1B gene located in the 2q22 region. We report here the first Moroccan patient, born to consanguineous parents, with Mowat-Wilson syndrome, due to a de novo, unreported mutation of the ZFHX1B gene.     

The expanding phenotypic spectrum of female <Emphasis Type="Italic">SLC9A6</Emphasis> mutation carriers: a case series and review of the literature

Christianson syndrome (OMIM 300243), caused by mutations in the X-linked SLC9A6 gene, is characterized by severe global developmental delay and intellectual disability, developmental regression, epilepsy, microcephaly and impaired ocular movements. It shares many common features with Angelman syndrome. Carrier females have been described as having learning difficulties with mild to moderate intellectual disability, behavioural issues and psychiatric illnesses. There is little literature on the carrier female phenotype of Christianson syndrome. We describe a large extended family with three affected males, four carrier females, one presumed carrier female and one obligate carrier female with a c.190G>T, p.E64X mutation known to cause a premature stop codon in SLC9A6. We characterize and expand the clinical phenotype of female SLC9A6 mutation carriers by comparing our described family with female carriers previously discussed in the literature. In particular, we highlight the neurodevelopmental and psychiatric phenotypes observed in our family and previous reports.     

Biochemical and clinical characteristics of creatine deficiency syndromes

Creatine deficiency syndromes are a newly described group of inborn errors of creatine synthesis (arginine:glycine amidinotransferase (AGAT) deficiency and guanidinoacetate methyltransferase (GAMT) deficiency) and of creatine transport (creatine transporter (CRTR) deficiency). The common clinical feature of creatine deficiency syndromes is mental retardation and epilepsy suggesting main involvement of cerebral gray matter. The typical biochemical abnormality of creatine deficiency syndromes is cerebral creatine deficiency, which is demonstrated by in vivo proton magnetic resonance spectroscopy. Measurement of guanidinoacetate in body fluids may discriminate between the GAMT (high concentration), AGAT (low concentration) and CRTR (normal concentration) deficiencies. Further biochemical characteristics include changes in creatine and creatinine concentrations in body fluids. GAMT and AGAT deficiency are treatable by oral creatine supplementation, while patients with CRTR deficiency do not respond to this type of treatment. The creatine deficiency syndromes are underdiagnosed, so their possibility should be considered in all children affected by unexplained mental retardation, seizures and speech delay.     

Two novel RAD21 mutations in patients with mild Cornelia de Lange syndrome-like presentation and report of the first familial case

Cornelia de Lange syndrome (CdLS) is a developmental disorder characterized by limb reduction defects, characteristic facial features and impaired cognitive development. Mutations in the NIPBL gene predominate; however, mutations in other cohesin complex genes have also been implicated, particularly in atypical and mild CdLS cases. Missense mutations and whole gene deletions in RAD21 have been identified in children with growth retardation, minor skeletal anomalies and facial features that overlap findings in individuals with CdLS. We report the first intragenic deletion and frameshift mutations identified in RAD21 in two patients presenting with atypical CdLS. One patient had an in-frame deletion of exon 13, while the second patient had a c.592_593dup frameshift mutation. The first patient presented with developmental delay, hypospadias, inguinal hernia and dysmorphic features while, the second patient presented with developmental delay, characteristic facial features, hirsutism, and hand and feet anomalies, with the first patient being milder than the second. The in-frame deletion mutation was found to be inherited from the mother who had a history of melanoma and other unspecified medical problems.     

De novo translocation (2;18)(q21;q22) in a child with severe epilepsy, developmental delay and mild dysmorphism

De novo translocation (2;18)(q21;q22) in a patient with severe epilepsy developmental delay and mild dysmorphism: We report on a patient presenting with severe epilepsy, hypotonia, developmental delay, blepharophimosis, low-set ears, camptodactyly and tapering fingers, and cutaneous syndactyly of toes II and III of the right foot. The MRI showed some loss of volume of the white matter and delayed myelination, no other specific anomalies were present. Chromosome analysis revealed a translocation involving chromosomes 2 and 18, which was characterized further by FISH using band-specific probes. The possibility of a submicroscopic deletion is discussed and the patient is compared with patients reported in the literature with either 2q21 or 18q22 deletion.     

X-linked myoclonus epilepsy explained as a maternally inherited mitochondrial disorder

A family with myoclonus epilepsy has been described previously as suffering from an X-linked disorder, because at least four males were affected, and only mild and variable symptoms were seen in some female carriers. In this family, we have now identified a mitochondrial AG (8344) heteroplasmic point mutation. This point mutation has been described in families with maternally inherited myoclonus epilepsy and ragged red fibers. The degree of severity of the disorder in the different family members was reflected in the relative quantity of mutated mitochondrial DNA. It is concluded that the mode of inheritance in this family is not X-linked but maternal.     

Identification, characterization and cloning of SLC6A8C, a novel splice variant of the creatine transporter gene

SLC6A8 deficiency is caused by mutations in the X-linked creatine transporter gene (SLC6A8), which leads to cerebral creatine deficiency, mental retardation, speech and language delay, autistic-like behaviour and epilepsy. Insight in the mechanism of how the transporter is regulated is largely unknown and it is of importance for the development of successful treatment strategies of cerebral creatine deficient syndromes. Our goal was to characterize CRT2 (SLC6A8B), a published splice variant of the creatine transporter. Surprisingly, using RT-PCR we found a novel splice variant, SLC6A8C, which is predominantly found in human tissues with a high energy requirement such as brain, kidney, heart, small intestines and skeletal muscle, where SLC6A8 transporter is most required. The 5' untranslated region (UTR) of the SLC6A8C mRNA was identified using the Smart Race cDNA amplification kit. The SLC6A8C mRNA contains intron 4 and exons 5 through 13 of SLC6A8, including part of the 3' UTR. An open reading frame was found, which predicts a truncated protein identical to the SLC6A8 transporter, comprising the five last C-terminal transmembrane domains of the SLC6A8 transporter. SLC6A8C open reading frame was cloned as a fusion protein with EGFP and the SLC6A8C protein expression was detected by Western Blot. RT-PCR and sequence analysis showed that this splice variant is conserved in evolution, since we also detected it in mouse. This study reveals the presence of a novel SLC6A8 splice variant, SLC6A8C in human and mouse.     

The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the alpha-chain of beta-hexosaminidase

The Ashkenazi Jewish population is enriched for carriers of a fatal form of Tay-Sachs disease, an inherited disorder caused by mutations in the alpha-chain of the lysosomal enzyme, beta-hexosaminidase A. Until recently it was presumed that Tay-Sachs patients from this ethnic isolate harbored the same alpha-chain mutation. This was disproved by identification of a splice junction defect in the alpha-chain of an Ashkenazi patient which could be found in only 20-30% of the Ashkenazi carriers tested. In this study we have isolated the alpha-chain gene from an Ashkenazi Jewish patient, GM515, with classic Tay-Sachs disease who was negative for the splice junction defect. Sequence analysis of the promoter region, exon and splice junctions regions, and polyadenylation signal area revealed a 4-base pair insertion in exon 11. This mutation introduces a premature termination signal in exon 11 which results in a deficiency of mRNA in Ashkenazi patients. A dot blot assay was developed to screen patients and heterozygote carriers for the insertion mutation. The lesion was found in approximately 70% of the carriers tested, thereby distinguishing it as the major defect underlying Tay-Sachs disease in the Ashkenazi Jewish population.     

X-linked creatine transporter (SLC6A8) mutations in about 1% of males with mental retardation of unknown etiology

Mutations in the creatine transporter gene, SLC6A8 (MIM 30036), located in Xq28, have been found in families with X-linked mental retardation (XLMR) as well as in males with idiopathic mental retardation (MR). In order to estimate the frequency of such mutations in the MR population, a screening of 478 males with MR of unknown cause was undertaken. All 13 exons of SLC6A8 were sequenced using genomic DNA. Six novel potentially pathogenic mutations were identified that were not encountered in at least 588 male control chromosomes: two deletions (p.Asn336del, p.Ile347del) and a splice site alteration (c.1016+2C>T) are considered pathogenic based on the nature of the variant. A mutation (p.Arg391Trp) should be considered pathogenic owing to its localization in a highly conserved region. Two other missense variants (p.Lys4Arg, p.Gly26Arg) are not conserved but were not observed in over 300 male control chromosomes. Their pathogenicity is uncertain. A missense variant (p.Val182Met), was classified as a polymorphism based on a normal creatine/creatinine (Cr:Crn) ratio and cerebral creatine signal in proton magnetic resonance spectroscopy (H-MRS) in the patient. Furthermore, we found 14 novel intronic and neutral variants that were not encountered in at least 280 male control chromosomes and should be considered as unclassified variants. Our findings of a minimum of four pathogenic mutations and two potentially pathogenic mutations indicate that about 1% of males with MR of unknown etiology might have a SLC6A8 mutation. Thus, DNA sequence analysis and/or a Cr:Crn urine screen is warranted in any male with MR of unknown cause.Amy J. Clark and Efraim H. Rosenberg have contributed equally to this work.