Phenotype and genotype in 101 males with X-linked creatine transporter deficiency.

BACKGROUND: Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype--genotype correlation has been lacking. METHODS: We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). RESULTS AND CONCLUSIONS: Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.

The management of pregnancy in maple syrup urine disease: experience with two patients.

We describe the management and outcomes of pregnancy in two women affected with Maple syrup urine disease (MSUD). Both patients had classical disease diagnosed in the newborn period and were managed with low-protein diets and supplements, although compliance was moderately poor throughout life. Both pregnancies were complicated by poor compliance and one patient had a metabolic decompensation, which included seizures and profound encephalopathy, at the end of the first trimester. Peri-partum management required a coordinated team approach including a high-calorie and low-protein diet. Both patients had elevated leucine levels in the post-partum period - one due to mastitis and the other due to poor dietary and supplement compliance combined with uterine involution. On later review, leucine had returned to pre-pregnancy levels. Both infants were unaffected and have made normal developmental progress in the subsequent 1 to 2 years.

The mild form of menkes disease: a 34 year progress report on the original case.

Classical Menkes disease is a neurodegenerative disorder caused by mutations in the copper-transporting ATPase ATP7A gene which, when untreated, is usually fatal in early childhood. A mild form of Menkes disease was originally reported in 1981 and clinical progress of the patient at 10 years described subsequently. The causative mutation is c.4085C>T in exon 21, causing an alanine to valine substitution in the highly conserved TM7 domain at the C-terminal end of the Menkes protein. Here we report his status at 34 years of age. Intellectual impairment is mild. Ataxia has nearly resolved but motor retardation, dysarthria and an extreme slow speech rate remain. In contrast to patients with the occipital horn syndrome, there have been no connective tissue complications of his mild Menkes disease. He has been under long-term copper therapy for more than 30 years and he continues to enjoy a good quality of life.

Mapping spatial and field dependence of magnetic domain memory by soft X-ray speckle metrology.

The occurrence of magnetic domain memory has been observed in ferromagnets, either induced by structural defects or by exchange couplings. Being able to quantify the amount of memory as a function of length scale, field and temperature is both of fundamental and technological importance. A technique has been refined to statistically quantify the magnetic domain memory in ferromagnetic thin films by using coherent soft-X-ray scattering metrology. This technique, based on cross-correlating magnetic speckle patterns, provides a unique way to map out the behavior of domain memory. Here, the details of our correlation method and the necessary treatment of the X-ray scattering images to extract spatial and field dependences in the memory information are reviewed. The resulting correlation maps, measured on [Co/Pd]IrMn multilayers, show how magnetic domain memory evolves at various spatial scales, as a function of the field magnitude throughout magnetization cycles, but also as a function of field cycling and of temperature. This technique can easily be applied to a wide variety of systems presenting memory effects, in soft and hard matter, and also to dynamical studies.

An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples.

BACKGROUND: Branched chain amino acid (BCAA) analysis is needed for the diagnosis and management of patients with maple syrup urine disease (MSUD). We report an improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of BCAAs and Allo-Ile in dried blood spot (DBS) samples. METHODS: BCAAs were extracted from a 3 mm blood spot into methanol/water containing stable isotope internal standards. Eluents were dried and reconstituted in the mobile phase. Gradient elution was performed on an Acquity™ BEH C(18) (100 × 2.1 mm, 1.7 µm) column. BCAAs were detected and quantified in the multiple reaction monitoring mode in a five-minute analysis. RESULTS: The assay was calibrated to give best possible alignment with plasma results. Retrospective analysis of newborn DBSs from six classic MSUD patients showed elevated alloisoleucine (Allo-Ile) in all cases. Two of four patients with mild disease had normal values; the other two had significant elevations in Allo-Ile. CONCLUSIONS: Analysis of BCAA in DBS by UPLC-MS/MS is a useful second tier newborn screening test to identify classical MSUD and for monitoring of remote patients.

3-Methylglutaconic aciduria type I redefined: a syndrome with late-onset leukoencephalopathy.

OBJECTIVE: 3-Methylglutaconic aciduria type I is a rare inborn error of leucine catabolism. It is thought to present in childhood with nonspecific symptoms; it was even speculated to be a nondisease. The natural course of disease is unknown. METHODS: This is a study on 10 patients with 3-methylglutaconic aciduria type I. We present the clinical, neuroradiologic, biochemical, and genetic details on 2 new adult-onset patients and follow-up data on 2 patients from the literature. RESULTS: Two unrelated patients with the characteristic biochemical findings of 3- methylglutaconic aciduria type I presented in adulthood with progressive ataxia. One patient additionally had optic atrophy, the other spasticity and dementia. Three novel mutations were found in conserved regions of the AUH gene. In both patients, MRI revealed extensive white matter disease. Follow-up MRI in a 10-year-old boy, who presented earlier with isolated febrile seizures, showed mild abnormalities in deep white matter. CONCLUSION: We define 3-methylglutaconic aciduria type I as an inborn error of metabolism with slowly progressive leukoencephalopathy clinically presenting in adulthood. In contrast to the nonspecific findings in pediatric cases, the clinical and neuroradiologic pattern in adult patients is highly characteristic. White matter abnormalities may already develop in the first decades of life. The variable features found in affected children may be coincidental. Long-term follow-up in children is essential to learn more about the natural course of this presumably slowly progressive disease. Dietary treatment with leucine restriction may be considered.

Contiguous gene deletion syndrome in a female with ornithine transcarbamylase deficiency.

OTC deficiency, a partially dominant X-linked trait, is the most frequent inborn error of the urea cycle. We describe a female patient with a contiguous gene deletion syndrome encompassing the OTC, DMD, RPGR, CYBB and XK genes, amongst others, only manifesting features of OTC deficiency. Molecular characterization was ascertained by MLPA and confirmed by CGH microarray, which revealed an 8.7 Mb deletion of the X-chromosome. Complete de novo deletion of the OTC gene led to a severe clinical phenotype in the proband. The application of high resolution molecular genetic techniques such as MLPA and array CGH, in mutation negative OTC cases allows the identification of chromosomal rearrangements, such as large deletions and provides information for accurate genetic counseling and prenatal diagnosis.

Glutaric aciduria type I: outcome following detection by newborn screening.

Glutaric aciduria type I (GA I), a cerebral organic acidaemia with the potential for severe neurological consequences, can now be detected by tandem mass spectrometry newborn screening. Early detection with implementation of careful management strategies appears to lessen the likelihood of neurological damage. We assessed the outcome in all 10 GA I patients detected in New South Wales during the last decade. Three patients were detected clinically and 7 by newborn screening. Diagnosis was confirmed by detection of significantly elevated urinary 3-hydroxybutyrate and glutarate in urine, isolated elevation of glutarylcarnitine in plasma, typical clinical and MRI findings in several, and mutation analysis or enzyme analysis on cultured skin fibroblasts in 4 cases. The birth frequency was 1:90,000. Following diagnosis, treatment was initiated in all children with oral carnitine (100 mg/kg per day) and a low-protein diet supplemented with a lysine-free, low-tryptophan amino acid formula. Disability was assessed in fields of motor, cognitive and speech development and scored according to Kyllerman. Clinically diagnosed patients were all symptomatic, with severity scores (out of 9) of 3, 5 and 9. Six of seven patients detected by newborn screening are asymptomatic, 4 being aged 2-6 years. One patient had a severe decompensation at 7 months, despite full management advice and treatment, and later died. Our data support previous findings that early diagnosis reduces neurological complications, but show that even with early diagnosis and careful management severe complications may ensue in some.

n of 1 trial for an ornithine transcarbamylase deficiency carrier.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked disorder of the urea cycle. It is often fatal in affected males. Treatment for affected individuals includes dietary protein restriction, activation of alternative pathways of nitrogen excretion and L-arginine supplementation. Depending on the amount of X chromosome inactivation skewing, females show variable clinical manifestations, and sometimes the need for treatment, including medications, is unclear. We conducted an n of 1 randomized controlled trial on an obligate OTC carrier. The treating physician and patient were blinded to treatment. Either placebo capsules or L-arginine capsules were given for weekly periods. Weekly efficacy indicators included plasma arginine and glutamine levels and a quality of life/mood assessment questionnaire scale. Clear evidence of benefit with L-arginine compared to placebo was shown. This is the first time an n of 1 randomized controlled trial has been reported for an X-linked metabolic condition. Despite some logistic hurdles, we have demonstrated that this method was an effective tool for determining the value of treatment. We propose that other rare metabolic conditions may be amenable to such trials, if the benefit of treatment is in doubt.

The consequences of extended newborn screening programmes: do we know who needs treatment?

The development of an evidence base for newborn screening is especially difficult because of the rarity of disorders now detectable. One consequence of expanded newborn screening is that physicians are being called upon to manage asymptomatic babies with persistent biochemical disturbances that indicate likely enzyme deficiencies. Some of these may be very mild. There is not always agreement as to who should be treated. Particular problems are seen with disorders that were previously thought very rare but are now found frequently by newborn screening. Some of these disorders appear benign or nearly so, and in the present state of knowledge should clearly not be included in routine newborn screening panels.

Recent advances in newborn screening.

The introduction of tandem mass spectrometry has unquestionably been the most striking recent advance in newborn screening. A single test is applied for the simultaneous diagnosis of a number of disorders, making it possible to screen for some disorders that might otherwise have seemed too rare. Current screening is for disorders of metabolism of amino acids, organic acids and fatty acids. Assay performance for detection of disorders appears very good, but rarity of disorders, varied definitions and systems for follow-up and lack of databases for inborn errors of metabolism diagnosed clinically means that there is as yet insufficient information about most disorders. The technology can be applied to a much wider range of compounds, and the field looks set to expand. A key feature of newborn screening programmes must be the assessment of outcomes, and a major reason for the lack of uniformity in the approach adopted in different countries is the paucity of information on this. The available evidence points to overall advantages flowing from early diagnosis by screening, with reduction in mortality and morbidity. More studies are clearly needed and some are under way. The next new group of disorders already proposed for newborn screening is the lysosomal storage disorders. Attitudes may be changing about what it is desirable to include in a newborn screening programme, and this will indeed pose new ethical dilemmas.

Guideline for the diagnosis and management of glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type I).

Glutaryl-CoA dehydrogenase (GCDH) deficiency is an autosomal recessive disease with an estimated overall prevalence of 1 in 100 000 newborns. Biochemically, the disease is characterized by accumulation of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutarylcarnitine, which can be detected by gas chromatography-mass spectrometry of organic acids or tandem mass spectrometry of acylcarnitines. Clinically, the disease course is usually determined by acute encephalopathic crises precipitated by infectious diseases, immunizations, and surgery during infancy or childhood. The characteristic neurological sequel is acute striatal injury and, subsequently, dystonia. During the last three decades attempts have been made to establish and optimize therapy for GCDH deficiency. Maintenance treatment consisting of a diet combined with oral supplementation of L: -carnitine, and an intensified emergency treatment during acute episodes of intercurrent illness have been applied to the majority of patients. This treatment strategy has significantly reduced the frequency of acute encephalopathic crises in early-diagnosed patients. Therefore, GCDH deficiency is now considered to be a treatable condition. However, significant differences exist in the diagnostic procedure and management of affected patients so that there is a wide variation of the outcome, in particular of pre-symptomatically diagnosed patients. At this time of rapid expansion of neonatal screening for GCDH deficiency, the major aim of this guideline is to re-assess the common practice and to formulate recommendations for diagnosis and management of GCDH deficiency based on the best available evidence.

Non-ketotic hyperglycinemia is usually not detectable by tandem mass spectrometry newborn screening.

Diagnosis of Non-Ketotic Hyperglycinemia by MSMS newborn screening might benefit patients with post-neonatal presentation. We screened 733,527 babies over eight years, and nine babies were subsequently diagnosed with NKHG. Two had newborn glycine levels above our cut-off and presented within 72 h. The remaining patients could not have been diagnosed by newborn screening without an unacceptably high recall rate. We conclude that babies with NKHG are not usually identifiable by current newborn screening strategies.

Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the GeNorm algorithm.

Eleven reference genes (18s ribosomal ribonucleic acid [RNA], 28s ribosomal RNA, ubiquitin, beta-actin, glycerine aldehyde dehydrogenase, ATP-synthase subunit 5B, hydroxymethyl-bilane synthase, hypoxanthine-phosphoribosyl transferase, ribosomal protein L32, tryptophan 5-monooxygenase activation protein (zeta polypeptide), and TATA-Box binding protein) were analyzed in use as references for gene expression profiling experiments using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in canine mammary tumors. The transcription level of the candidates was measured in 22 histologically characterized excised tumor specimens from mammary gland tissue and 22 samples of non-neoplastic mammary tissue samples from the same individuals. Results were used to rank candidate reference genes using the GeNorm tool. It was determined that in samples of canine mammary gland tissue, a combination of hypoxanthine-phosphoribosyl transferase, ATP-synthase subunit 5B, ribosomal protein L32 and ubiquitin yields stable reference gene expression levels, whereas the use of glycerin aldehyde dehydrogenase or ribosomal RNA is unsuitable for normalization of qRT-PCR results in this tissue type.

Newborn screening may fail to identify intermediate forms of maple syrup urine disease.

The New South Wales state-wide newborn screening programme has offered comprehensive screening for inborn errors of metabolism, including MSUD, using electrospray tandem mass spectrometry since 1998. Over this period, a number of patients with classic MSUD have been identified with subsequent good neurological outcome. We describe two patients with an intermediate form of MSUD who presented later in childhood. Retrospective review of their newborn screening results demonstrates that the diagnosis could not have been made by current newborn screening. Their neurological outcome is much less satisfactory. Despite the usefulness of expanded newborn screening programmes in detecting severe neonatal presentations of inborn errors of metabolism, partial enzyme deficiencies may not be detected. Metabolic diseases still need to be considered in appropriate clinical situations later in life.

Clinical approach to inborn errors of metabolism presenting in the newborn period.

Inborn errors of metabolism are individually rare, but collectively are responsible for significant levels of paediatric morbidity and mortality. More than 400 biochemically diverse inborn errors of metabolism have been identified. Recent advances in the diagnosis and treatment of these disorders have substantially improved the prognosis for many of them. Paediatricians and neonatologists play a vital role in identifying which patients need to be investigated. The diagnosis of an inborn error of metabolism often needs to be established quickly in order to prevent death or permanent neurological sequelae, and this should be carried out in collaboration with a specialized unit. The present review provides a practical approach to the recognition and investigation of neonates in whom an inborn error may be present. We also provide guidelines for the stabilization and initial management of infants at high risk of a metabolic disorder.

Anorexia nervosa (restrictive subtype) is associated with a polymorphism in the novel norepinephrine transporter gene promoter polymorphic region.

Long-term weight-restored patients with anorexia nervosa (AN) have lower norepinephrine levels than controls. Since this may reflect altered reuptake by the norepinephrine transporter (NET), we hypothesised that the NET gene was involved in the genetic component of AN. PCR-amplification of an AAGG repeat island (AAGG1) in the NET gene promoter region revealed a novel 343-bp sequence with five additional AAGG repeat islands (AAGG2-AAGG6). We named the sequence from AAGG1 to AAGG6 inclusive, the NET gene promoter polymorphic region (NETpPR). A 4-bp deletion (S4) or insertion (L4) in AAGG4 resulted in the net loss or gain, respectively, of a putative Elk-1 transcription factor site. The transmission disequilibrium test(TDT) with 87 Australian trios (patient plus parents) demonstrated significant preferential transmission of L4 (McNemar's chi(2) = 7.806, df = 1, P = 0.0052, odds ratio: 2.1) from parent to child with restricting AN (AN-R), suggesting that L4 or a DNA variant in linkage disequilibrium with it, doubles the risk for developing AN-R.

3-Hydroxyglutarate excretion is increased in ketotic patients: implications for glutaryl-CoA dehydrogenase deficiency testing.

Three patients with ketosis had increased excretion of 3-hydroxyglutarate (21.8-37.9 micromol/mmol creatinine; controls 2.3 +/- 1.6), an indicator of glutaryl-CoA dehydrogenase deficiency (GDHD), which normalized when the patients were nonketotic. Clinical assessment of all three patients and enzyme studies in one patient were not consistent with GDHD. These findings were compared with those of other ketotic patients, who showed statistically significant increases in 3-hydroxyglutarate excretion (9.4 +/- 5.0 micromol/mmol creatinine; p < 0.01), and with those of a child with confirmed GDHD when she was both ketotic and nonketotic. Secondary increase in 3-hydroxyglutarate excretion during ketosis is a potential confounder in the diagnosis of GDHD.

Hartnup disorder: polymorphisms identified in the neutral amino acid transporter SLC1A5.

Hartnup disorder is an inborn error of renal and gastrointestinal neutral amino acid transport. The cloning and functional characterization of the 'system B0' neutral amino acid transporter SLC1A5 led to it being proposed as a candidate gene for Hartnup disorder. Linkage analysis performed at 19q13.3, the chromosomal position of SLC1A5, was suggestive of an association with the Hartnup phenotype in some families. However, SLC1A5 was not linked to the Hartnup phenotype in other families. Linkage analysis also excluded an alternative candidate region at 11q13 implicated by a putative mouse model for Hartnup disorder. Sequencing of the coding region of SLC1A5 in Hartnup patients revealed two coding region polymorphisms. These mutations did not alter the predicted amino acid sequence of SLC1A5 and were considered unlikely to play a role in Hartnup disorder. There were no mutations in splice sites flanking each exon. Quantitative RT-PCR of SLC1A5 messenger RNA in affected and unaffected subjects did not support systemic differences in expression as an explanation for Hartnup disorder. In the six unrelated Hartnup pedigrees studied, examination of linkage at 19q13.3, polymorphisms in the coding sequence and quantitation of expression of SLC1A5 did not suffice to explain the defect in neutral amino acid transport.