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.

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.

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.

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.

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.

The pathogenesis of coronary artery disease. A possible role for methionine metabolism.

Homocystinuria, an abnormality of methionine metabolism is associated with severe vascular disease in infancy and childhood. Homocysteine is formed during the metabolism of methionine and accumulations of this and of cysteine-homocysteine mixed disulfide in the plasma indicate a partial block in the methionine degradation pathway. Methionine metabolism was investigated in 25 patients aged under 50 with angiographically proved coronary artery disease and in 22 control patients, of whom 17 had normal coronary arteries at angiography and 5 were healthy volunteers. After an overnight fast, venous blood was drawn before and 4 h after oral L-methionine, 100 mg/kg. Plasma methionine levels at 4 h were not different in the two groups, but there were significant differences in the levels of cysteine-homocysteine mixed disulfide. This was detected in 5 of 22 in the noncoronary group and in higher concentration in 17 of 25 coronary patients (P less than 0-01). Age, weight, height, body-mass index, glucose tolerance, fasting serum urate, and triglycerides were not different, but serum cholesterol was higher in the coronary patients (P lessthan 0.01). These results suggest a reduced ability to metabolise homocysteine in some patients with premature coronary artery disease when this pathway is stressed.

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.

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.

Vascular outcome in patients with homocystinuria due to cystathionine beta-synthase deficiency treated chronically: a multicenter observational study.

An inborn error of metabolism, homocystinuria due to cystathionine beta-synthase deficiency, results in markedly elevated levels of circulating homocysteine. Premature vascular events are the main life-threatening complication. Half of all untreated patients have a vascular event by 30 years of age. We performed a multicenter observational study to assess the effectiveness of long-term homocysteine-lowering treatment in reducing vascular risk in 158 patients. Vascular outcomes were analyzed and effectiveness of treatment in reducing vascular risk was evaluated by comparison of actual to predicted number of vascular events, with the use of historical controls from a landmark study of 629 untreated patients with cystathionine beta-synthase deficiency. The 158 patients had a mean (range) age of 29.4 (4.5 to 70) years; 57 (36%) were more than 30 years old, and 10 (6%) were older than 50 years. There were 2822 patient-years of treatment, with an average of 17.9 years per patient. Plasma homocysteine levels were markedly reduced from pretreatment levels but usually remained moderately elevated. There were 17 vascular events in 12 patients at a mean (range) age of 42.5 (18 to 67) years: pulmonary embolism (n=3), myocardial infarction (n=2), deep venous thrombosis (n=5), cerebrovascular accident (n=3), transient ischemic attack (n=1), sagittal sinus thrombosis (n=1), and abdominal aortic aneurysm (n=2). Without treatment, 112 vascular events would have been expected, for a relative risk of 0.09 (95% CI 0.036 to 0.228; P<0.0001). Treatment regimens designed to lower plasma homocysteine significantly reduce cardiovascular risk in cystathionine beta-synthase deficiency despite imperfect biochemical control. These findings may be relevant to the significance of mild hyperhomocysteinemia that is commonly found in patients with vascular disease.

First prenatal diagnosis of the carnitine transporter defect.

We report the first attempt at prenatal diagnosis of the carnitine transporter defect in a fetus at high risk of having the disorder. Analysis of cultured CVS after prolonged culture predicted that the fetus was not affected but might be heterozygous for the carnitine transporter defect, but chromosome 15 satellite DNA markers showed no paternal contribution, suggesting that the CVS cells assayed were of predominantly maternal origin. Subsequent assay of cultured amniocytes predicted that the fetus would be affected, and this was confirmed in the newborn period. We conclude that prenatal diagnosis of the carnitine transporter defect is possible, but where results depend on extended culture of CVS, molecular studies should be performed to confirm genetic contributions from both parents.

B vitamins and homocysteine in cardiovascular disease and aging.

The sulfur-containing amino acid, homocysteine, is formed from the essential amino acid methionine, and a number of B vitamins are involved in methionine metabolism. Pyridoxine, vitamin B6, is a cofactor for cystathionine beta synthase, which mediates the transformation of homocysteine to cystathionine, the initial step in the transsulfuration pathway and the urinary excretion of sulfur. In a normal diet there is conservation of the carbon skeleton, and about 50% of the homocysteine formed is remethylated to methionine via steps that require folic acid and vitamin B12. A deficiency of any of these three vitamins leads to modest homocyst(e)ine elevation, as does diminished renal function, both of which are common in the elderly. It is also established that homocyst(e)ine elevation of this order is associated with increased cardiovascular risk but is also associated with most established risk factors, although it is thought to be an independent contributor. In the inborn error of metabolism homocystinuria due to cystathionine beta synthase deficiency there is greatly increased circulating homocyst(e)ine and a clear association with precocious vascular disease. In about 50% of these patients there is a vascular event before the age of 30 years. The homocysteine-induced adverse vascular changes appear to result from endothelial and smooth muscle cell effects and increased thrombogenesis. We have documented a highly significant reduction in the occurrence of vascular events during 539 patient years of treatment in 32 patients with cystathionine beta synthase deficiency (mean age 30 years, range 9-66 years) by aggressive homocyst(e)ine lowering with pyridoxine, folic acid, and B12 (p = 0.0001). The 15 pyridoxine nonresponsive patients also received oral betaine. Although a cause and effect relationship is postulated for the increased cardiovascular risk associated with mild homocysteine elevation, a common cause of this elevation is the methylenetetrahydrofolate reductase C677T mutation. Homozygotes occur in about 11% of Caucasian populations. However, the mutation is not associated with increased coronary risk. Since mild homocysteine elevation is easily normalized by B vitamin supplementation, usually with folic acid, it remains for controlled clinical trials of this inexpensive therapy to determine whether normalizing mild homocyst(e)ine elevation reduces cardiovascular risk.

Methionine transamination in patients with homocystinuria due to cystathionine beta-synthase deficiency.

To assess the ability of patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency to perform the reactions of the methionine transamination pathway, the concentrations of the products of this pathway were measured in plasma and urine. The results clearly demonstrate that CBS-deficient patients develop elevations of these metabolites once a threshold near 350 micromol/L for the concurrent plasma methionine concentration is exceeded. The absence of elevated methionine transamination products previously reported among 16 CBS-deficient B6-responsive patients may now be attributed to the fact that in those patients the plasma methionine concentrations were below this threshold. The observed elevations of transamination products were similar to those observed among patients with isolated hypermethioninemia. Plasma homocyst(e)ine did not exert a consistent effect on transamination metabolites, and betaine appeared to effect transamination chiefly by its tendency to elevate methionine. Even during betaine administration, the transamination pathway does not appear to be a quantitatively major route for the disposal of methionine.

Prevalence of unsuspected urinary bacterial contamination: effects of screening tests for detection of inborn errors of metabolism.

Hospitals and doctors submitting urine samples to a central reference laboratory screening for inborn errors of metabolism had been given precise instructions on sample handling designed to prevent growth of contaminating bacteria. 140 consecutive samples were examined bacteriologically and 22% had a heavy growth of bacteria (greater than 10(5) organisms/ml). Contamination was commonest in samples from children under 2 years. Normal human urine samples spiked with various amino acids were inoculated with pure cultures of different urinary pathogens and then incubated, with control samples, at 37 degrees C. Urinary amino acids were measured either semi-quantitatively by high-voltage electrophoresis or quantitatively by ion-exchange chromatography. Very wide-ranging changes in amino acid levels were noted in some inoculated samples after incubation. In two urine samples spiked with 8 mg/ml arginine, this had disappeared after 24-48 h. Unsuspected urinary bacterial contamination may be a more important source of false positive and false negative results in the investigation of disorders of amino-acid metabolism than previously supposed.

gamma-Glutamylglutamine identified in plasma and cerebrospinal fluid from hyperammonaemic patients.

gamma-Glutamylglutamine has been identified in plasma and cerebrospinal fluid. Preparative high voltage electrophoresis was used to isolate the compound from two to three ml of sample. Analysis of archival material from eight patients with urea cycle disorders showed that plasma gamma-glutamylglutamine was 20-214 mumol/l when the plasma glutamine was greater than 1,000 mumol/l and that the relationship was strongly positive (P less than 0.0005). Plasma gamma-glutamylglutamine concentration was also raised in one patient with secondary hyperammonaemia, but not in three other cases, one of whom had a plasma glutamine of 10,000 mumol/l. Cerebrospinal fluid from the last patient contained 97 mumol/l of gamma-glutamylglutamine. Samples of cerebrospinal fluid from two patients with urea cycle disorders were available and the gamma-glutamylglutamine levels were 203 and 313 mumol/l. gamma-Glutamylglutamine and 5-oxoproline concentrations were not significantly increased in urine from any of these patients.

Studies on a child suspected of having a dficiency in 3-hydroxy-3-methylglutaryl-Co A lyase.

The urine of a child who presented with hyperammonemia was found to contain elevated levels of 3-hydroxy-3-methylglutaric acid, 3-methylglutaconic acid and 3-methylglutaric acid. An increased excretion of these organic acids has been reported previously in a child with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Enzyme studies using cultured fibroblasts from this patient, however, indicated that the 3-hydroxy-3-methylglutaryl-CoA lyase activity was not markedly reduced.

Dicarboxylic aciduria: the response to fasting.

The urine of a child who presented with an episode of a disease resembling Reye's syndrome was found to contain large quantities of the dicarboxylic acids adipic and suberic acids, as well as the glycine conjugate of suberic acid, suberyl glycine. A variety of other dicarboxylic acids, both saturated and unsaturated, were also found in the urine at the time of the attack. It was found that the excretion of these unusual metabolites could be markedly increased by fasting for periods of greater than 10 h. These results indicate that the patient may have a defect in fatty acid oxidation which becomes clinically significant during periods of prolonged fasting.

Sweat testing following newborn screening for cystic fibrosis.

Sweat testing remains the "gold standard" for the diagnosis of cystic fibrosis (CF) and is a critical component of newborn screening programs. We retrospectively reviewed sweat test results reported to a neonatal screening program for CF with respect to completeness of reported results and the values recorded for sweat chloride (Cl(-)) and sodium (Na(+)) concentrations and the Cl(-):Na(+) ratio in screened infants. Thirty-nine of 85 DeltaF508 homozygous (DeltaF508/DeltaF508) and 270 of 274 DeltaF508 heterozygous (DeltaF508/-) infants had sweat tests reported to the screening program. Of those, 30 and 213 sweat test reports, respectively, were complete, i.e., sweat weight, sweat chloride, and sodium were reported. Three centers accounted for 37 of 68 (54%) incomplete results, and 4 centers performed 4 or less post-screening sweat tests in the study period. There were 6 DeltaF508 heterozygous infants with sweat Cl(-) concentrations of 40-60 mmol/L and 4 had CF confirmed by additional genotyping (n = 2) or clinical and repeat sweat Cl results (n = 2). Forty-one percent of DeltaF508/-infants with sweat Cl(-) <40 mmol/L had Cl:Na >1. We conclude that the reporting of incomplete sweat tests is common following newborn screening for CF. Infants with sweat Cl(-) levels of 40-60 mmol/L require further investigation and review, but they almost certainly have CF. The Cl(-):Na(+) ratio does not appear useful in establishing a diagnosis of CF in infants.

Reproductive decisions after neonatal screening identifies cystic fibrosis.

AIMS: To document the reproductive choices made by women in New South Wales, Australia, after neonatal screening has identified cystic fibrosis (CF). METHODS: A sample of women attending cystic fibrosis clinics in New South Wales who had a child (or children) diagnosed by neonatal screening between 1981 and 1996 were interviewed. RESULTS: Two thirds of the women chose to avoid having another child with CF. The uptake of prenatal diagnosis was 66% in women who had a subsequent pregnancy; of these 69% terminated or would have terminated an affected fetus. Fifty nine per cent of the women who decided against a further pregnancy made this decision in order to avoid having another child with CF. CONCLUSIONS: These data show that having a child with CF influenced subsequent reproductive choices. In addition to the medical advantages of an early diagnosis offered by neonatal screening, this also allows informed future reproductive decisions.