Quantification of N-acetyl-l-aspartate in dried blood spots: A simple and fast LC-MS/MS neonatal screening method for the diagnosis of Canavan disease.

BACKGROUND: Canavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene (ASPA). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective. METHODS: We therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/MS)-based method for quantification of N-acetylaspartate in dried blood spots and established reference ranges for neonates and older controls. With this test, we investigated 45 samples of 25 Canavan patients including 8 with a neonatal sample. RESULTS: Measuring N-acetylaspartate concentration in dried blood with this novel test, all Canavan patients (with variable severity) were well separated from the control group (median; range: 5.7; 1.6-13.6 µmol/L [n = 45] vs 0.44; 0.24-0.99 µmol/L [n = 59] (p < 0.05)). There was also no overlap when comparing neonatal samples of Canavan patients (7.3; 5.1-9.9 µmol/L [n = 8]) and neonatal controls (0.93; 0.4-1.8 µmol/L [n = 784]) (p < 0.05). CONCLUSIONS: We have developed a new LC-MS/MS-based screening test for early postnatal diagnosis of Canavan disease that should be further evaluated in a population-based study once a promising treatment becomes available. The method meets the general requirements of newborn screening and should be appropriate for multiplexing with other screening approaches that combine chromatographic and mass spectrometry techniques.

[Genetic analysis and prenatal diagnosis for a Chinese pedigree affected with Canavan disease].

OBJECTIVE: To explore the genetic basis for a Chinese patient suspected for Canavan disease. METHODS: Whole exome sequencing (WES) was carried out for the proband, and candidate variants were verified by Sanger sequencing of the proband, her parents and brother. Prenatal diagnosis was provided to her mother by chorionic villi sampling (CVS) upon her subsequent pregnancy. RESULTS: The proband, a 4-month-old female infant, had manifested drowsiness, hypotonia and apathy. Urine metabolism screening showed elevated N-acetylaspartic acid. Cranial magnetic resonance imaging revealed abnormal myelination and multiple abnormal signals in large brain areas. WES revealed that the proband has harbored compound heterozygous variants of the ASPA gene, namely c.187A>G (p.Arg63Gly) in exon 1 and c.634+1G>A (P.?) in exon 4. Sanger sequencing confirmed that the c.187A>G (p.Arg63Gly) and c.634+1G>A (p.?) variants were respectively inherited from her mother and father. Her phenotypically normal brother has carried a heterozygous c.634+1G>A (p.?) variant. Prenatal diagnosis by CVS indicated that the fetus was a heterozygous carrier of the c.187A>G variant. CONCLUSION: WES can facilitate the diagnosis of Canavan disease, particularly for those lacking specific phenotypes of the disease. The compound heterozygous variants of the ASPA gene probably underlay the Canavan disease in this patient, and the result has enabled prenatal diagnosis for this family.

A case of juvenile Canavan disease with distinct pons involvement.

BACKGROUND: Canavan disease is a genetic neurodegenerative leukodystrophy that results in the spongy degeneration of the white matter. Its key clinical features in the infantile form are developmental delay, visual problems and macrocephaly. Congenital and juvenile forms have also been described. PATIENT DESCRIPTION: We report on a 13-year-old boy who is a high school student in a public school. He was diagnosed with juvenile Canavan disease, presenting with intentional tremor as the only clinical finding. RESULTS: Magnetic resonance imaging revealed mainly the involvement of the caudate nucleus and pons extending to the mesencephalon and also the putamen and the thalamus, with no apparent signal increase in the cerebral white matter. A homozygous p.Gly274Arg (c.820A>G) missense mutation was identified. CONCLUSION: Juvenile Canavan disease with mainly pons involvement has not been published before. Pons, caudate nucleus and basal ganglia involvement without any white matter being involved could be expected in juvenile Canavan disease as a rare form of the disease.

Next generation sequencing of RNA reveals novel targets of resveratrol with possible implications for Canavan disease.

Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondrial function, and more recently it was shown to restore fatty acid ß-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity. Many of RSV's beneficial effects are mediated by the transcriptional coactivator PGC-1α, a direct target of SIRT1 and a master regulator of the mitochondrial fatty acid oxidation. Despite numerous studies RSV's mechanism of action is still not completely elucidated. Our aim was to investigate the effects of RSV on gene regulation on a wide scale, possibly to detect novel genes whose up-regulation by RSV may be of interest with respect to disease treatment. We performed Next Generation Sequencing of RNA on normal fibroblasts treated with RSV. To investigate whether the effects of RSV are mediated through SIRT1 we expanded the analysis to include SIRT1-knockdown fibroblasts. We identified the aspartoacylase (ASPA) gene, mutated in Canavan disease, to be strongly up-regulated by RSV in several cell lines, including Canavan disease fibroblasts. We further link RSV to the up-regulation of other genes involved in myelination including the glial specific transcription factors POU3F1, POU3F2, and myelin basic protein (MBP). We also observe a strong up-regulation by RSV of the riboflavin transporter gene SLC52a1. Mutations in SLC52a1 cause transient multiple acyl-CoA dehydrogenase deficiency (MADD). Our analysis of alternative splicing identified novel metabolically important genes affected by RSV, among which is particularly interesting the α subunit of the stimulatory G protein (Gsα), which regulates the cellular levels of cAMP through adenylyl cyclase. We conclude that in fibroblasts RSV stimulates the PGC-1α and p53 pathways, and up-regulates genes affecting the glucose metabolism, mitochondrial ß-oxidation, and mitochondrial biogenesis. We further confirm that RSV might be a relevant treatment in the correction of FAO deficiencies and we suggest that treatment in other metabolic disorders including Canavan disease and MADD might be also beneficial.

Clinically Distinct Phenotypes of Canavan Disease Correlate with Residual Aspartoacylase Enzyme Activity.

We describe 14 patients with 12 novel missense mutations in ASPA, the gene causing Canavan disease (CD). We developed a method to study the effect of these 12 variants on the function of aspartoacylase-the hydrolysis of N-acetyl-l-aspartic acid (NAA) to aspartate and acetate. The wild-type ASPA open reading frame (ORF) and the ORFs containing each of the variants were transfected into HEK293 cells. Enzyme activity was determined by incubating cell lysates with NAA and measuring the released aspartic acid by LC-MS/MS. Clinical data were obtained for 11 patients by means of questionnaires. Four patients presented with a non-typical clinical picture or with the milder form of CD, whereas seven presented with severe CD. The mutations found in the mild patients corresponded to the variants with the highest residual enzyme activities, suggesting that this assay can help evaluate unknown variants found in patients with atypical presentation. We have detected a correlation between clinical presentation, enzyme activity, and genotype for CD.

A case of Canavan disease with microcephaly.

BACKGROUND: Canavan disease is an autosomal recessive disorder with spongy degeneration of white matter of the brain. It presents with developmental delay, visual problems and macrocephaly. PATIENT DESCRIPTION: We report a ten-month old boy with Canavan disease who presented with global developmental delay, seizures, abnormal eye movements and microcephaly. RESULTS: MRI brain revealed diffuse involvement of the supra tentorial white matter, globus pallidi, thalami, dentate nuclei and brainstem with sparing of the corpus callosum. The genetic testing revealed homozygous mutation of aspartoacylase gene [c.859 G>A (p.Ala287Thr)] in Exon 6. CONCLUSION: Possibility of Canavan disease should be considered even in the presence of microcephaly.

Atypical clinical and radiological course of a patient with Canavan disease.

Canavan disease (CD) is a rare metabolic disorder caused by aspartoacylase (ASPA) deficiency. It leads to severe neurological degeneration with spongiform brain degeneration. Accumulation of N-acetylaspartate (NAA) in brain and urine is specific to the disease and guides diagnosis. Magnetic resonance imaging (MRI) usually shows diffuse white matter abnormalities with involvement of the basal ganglia. Mild forms of the disease with a more favorable clinical course and radiological involvement of the basal ganglia without white matter abnormalities have also been reported. Here we report an atypical case of a girl aged nine years with CD. The disease started at the classical age of five months. Classical elevation of NAA in brain and urine was present and genetic analysis identified mutations in the ASPA gene. However, clinical evolution was milder than typical CD, with partial motor impairment and relatively well-preserved cognitive skills. MRI was also atypical with low white matter involvement and unusual topography and evolution of abnormalities in the basal ganglia.

Early diagnosis of Canavan syndrome: how can we get there?

Canavan syndrome is a rare genetic disorder characterised by progressive severe leukodystrophy involving the degeneration of white matter. Currently, there is no effective therapy, but after recent studies using early gene therapy, the outcome has appeared to improve. It is of fundamental importance to recognise signs of neonatal Canavan syndrome early on. We describe a case of neonatal Canavan syndrome in which diagnosis was made only at the fourth month of age.

Leukodystrophy with multiple beaded periventricular cysts: unusual cranial MRI results in Canavan disease.

A 3-year-old boy was admitted with psychomotor delay, spasticity, progressive visual loss, nystagmus, macrocephaly, and epileptic seizures for diagnostics. Cranial magnetic resonance imaging (MRI) revealed leukodystrophy and multicystic changes. Urine excretion of N-acetylaspartic acid was grossly increased, suggesting Canavan disease. Mutation screening of the ASPA gene confirmed this diagnosis. The underlying enzymatic defect causes accumulation of N-acetylaspartic acid and subsequent progressive myelin degeneration with characteristic spongy degeneration of the subcortical white matter, normally only seen histologically. We describe this case to show that spongy degeneration in Canavan disease may also be present macroscopically in the form of multiple beaded periventricular cysts on cranial MRI.

Canavan disease: an Arab scenario.

The autosomal recessive Canavan disease (CD) is a neurological disorder that begins in infancy. CD is caused by mutations in the gene encoding the ASPA enzyme. It has been reported with high frequency in patients with Jewish ancestry, and with low frequency in non-Jewish patients. This review will shed light on some updates regarding CD prevalence and causative mutations across the Arab World. CD was reported in several Arab countries such as Saudi Arabia, Egypt, Jordan, Yemen, Kuwait, and Tunisia. The population with the highest risk is in Saudi Arabia due the prevalent consanguineous marriage culture. In several studies, four novel mutations were found among Arabian CD patients, including two missense mutations (p.C152R, p.C152W), a 3346bp deletion leading to the removal of exon 3 of the ASPA gene, and an insertion mutation (698insC). Other previously reported mutations, which led to damage in the ASPA enzyme activities found among CD Arab patients are c.530 T>C (p.I177T), c.79G>A (p.G27R), IVS4+1G>T, and a 92kb deletion, which is 7.16kb upstream from the ASPA start site. This review will help in developing customized molecular diagnostic approaches and promoting CD carrier screening in the Arab world in areas where consanguineous marriage is common particularly within Saudi Arabia.

Canavan disease - unusual imaging features in a child with mild clinical presentation.

Canavan disease is a rare hereditary leukodystrophy that manifests in early childhood. Associated with rapidly progressive clinical deterioration, it usually results in death by the third year of life. The predominant MRI appearance is diffuse and symmetrical white matter disease. We discuss an atypical, late presentation of Canavan disease with a benign clinical course and uncharacteristic imaging features. This case introduces a previously unreported pattern of diffuse cortical abnormality without significant white matter involvement.

An atypical case of Canavan disease with stroke-like presentation.

BACKGROUND: Canavan disease is an autosomal recessive leukodystrophy caused by a deficiency of aspartoacylase. The disease has a severe course, with death occurring in the first few years of life. Atypical patients with mild courses have been reported, but acute presentations similar to stroke have not been well described. PATIENT DESCRIPTION: We present a boy who presented at 4 months of age with seizures after an episode of cardiopulmonary arrest is discussed. RESULTS: He was initially thought to have an ischemic watershed stroke based on his initial clinical presentation and magnetic resonance imaging. However, biochemical and follow-up radiologic evaluation were consistent with mild Canavan disease. DNA sequencing of the ASPA gene indicated one known mutation (A305E) and a novel mutation, L30V. Follow-up magnetic resonance imaging did not reveal the atrophy which would have been expected with watershed ischemia. Magnetic resonance spectroscopy demonstrated elevated N-acetyl aspartate to creatinine and N-acetyl aspartate to choline ratios. At 4 years of age, he was normocephalic, with mild clumsiness, speech delay, and seizures. CONCLUSIONS: This child's unusual acute presentation, along with his prolonged mild course, raises questions about the relationship between biochemical signs of abnormal aspartoacylase function and clinical findings. This patient highlights the need for long-term clinical follow-up of children with mild Canavan disease to clarify the significance of these biochemical abnormalities.

Canavan disease: clinical features and recent advances in research.

Canavan disease (CD) is a genetic neurodegenerative leukodystrophy that results in the spongy degeneration of white matter in the brain. CD is characterized by mutations in the gene encoding aspartoacylase (ASPA), the substrate enzyme that hydrolyzes N-acetylaspartic acid (NAA) to acetate and aspartate. Elevated NAA and subsequent deficiency in acetate associated with this disease cause progressive neurological symptoms, such as macrocephaly, visuocognitive dysfunction, and psychomotor delay. The prevalence of CD is higher among Ashkenazi Jewish people, and several types of mutations have been reported in the gene coding ASPA. Highly elevated NAA is more specific to CD than other leukodystrophies, and an examination of urinary NAA concentration is useful for diagnosing CD. Many researchers are now examining the mechanisms responsible for white matter degeneration or dysmyelination in CD using mouse models, and several persuasive hypotheses have been suggested for the pathophysiology of CD. One is that NAA serves as a water pump; consequently, a disorder in NAA catabolism leads to astrocytic edema. Another hypothesis is that the hydrolyzation of NAA in oligodendrocytes is essential for myelin synthesis through the supply of acetate. Although there is currently no curative therapy for CD, dietary supplements are candidates that may retard the progression of the symptoms associated with CD. Furthermore, gene therapies using viral vectors have been investigated using rat models. These therapies have been found to be tolerable with no severe long-term adverse effects, reduce the elevated NAA in the brain, and may be applied to humans in the future.

Four-and-one-half years' experience in monitoring of reproducibility of an MR spectroscopy system--application of in vitro results to interpretation of in vivo data.

The primary purpose of this work was to assess long-term in vitro reproducibility of metabolite levels measured using 1H MRS (proton magnetic resonance spectroscopy). The secondary purpose was to use the in vitro results for interpretation of 1H MRS in vivo spectra acquired from patients diagnosed with Canavan disease. 1H MRS measurements were performed in the period from April 2006 to September 2010. 118 short and 116 long echo spectra were acquired from a stable phantom during this period. Change-point analysis of the in vitro N-acetylaspartate levels was exploited in the computation of fT factor (ratio of the actual to the reference N-acetylaspartate level normalized by the reciprocity principle). This coefficient was utilized in the interpretation of in vivo spectra analyzed using absolute reference technique. The monitored time period was divided into six time intervals based on short echo in vitro data (seven time intervals based on long echo in vitro data) characterized by fT coefficient ranging from 0.97 to 1.09 (based on short echo data) and from 1.0 to 1.11 (based on long echo data). Application of this coefficient to interpretation of in vivo spectra confirmed increased N-acetylaspartate level in Canavan disease. Long-term monitoring of an MRS system reproducibility, allowing for absolute referencing of metabolite levels, facilitates interpretation of metabolic changes in white matter disorders.

Results of the College of American Pathology/American College of Medical Genetics and Genomics external proficiency testing from 2006 to 2013 for three conditions prevalent in the Ashkenazi Jewish population.

PURPOSE: The purpose of this study was to determine analytic performance of laboratories offering molecular testing for conditions such as Tay-Sachs disease, Canavan disease, and familial dysautonomia, which are prevalent in the Ashkenazi Jewish population. METHODS: The College of American Pathologists and the American College of Medical Genetics and Genomics cosponsor molecular proficiency testing for these disorders. Responses from 2006 to 2013 were analyzed for accuracy (genotyping and interpretations). RESULTS: Between 11 and 36 laboratories participated in each Tay-Sachs disease distribution. Samples tested per month were constant (2,900) from 2006 to 2011 but recently increased. Participants reporting <10 samples tested per month had longer turnaround times (42 vs. 7%, longer than 14 days; P = 0.03). Analytic sensitivity and specificity for US participants were 97.2% (95% confidence interval: 94.7-98.7%) and 99.8% (95% confidence interval: 99.1-99.9%), respectively. Of 11 genotyping errors, 2 were due to sample mix-up. Analytic interpretations were correct in 99.3% of challenges (956/963; 95% confidence interval: 98.5-99.7%). Better performance was found for Canavan disease and familial dysautonomia. International laboratories performed equally well. CONCLUSION: These results demonstrated high analytic sensitivity and specificity along with excellent analytic interpretation performance, confirming the genetics community impression that laboratories provide accurate test results in both diagnostic and screening settings. Proficiency testing can identify potential laboratory issues and helps document overall laboratory performance.

[Megalencephaly with dystonia revealing Canavan disease]. / Macrocéphalie avec dystonie révélant une maladie de Canavan.

Canavan disease, or N-acetyl aspartic aciduria, is an autosomal recessive leukodystrophy characterized by spongy degeneration of the brain. The disease results from the accumulation of N-acetyl aspartic acid in the brain, due to aspartoacylase deficiency. We report the case of a 6-month-old girl who presented with megalencephaly, peripheral hypertonia, and a developmental delay noticeable after 4 months of age. Magnetic resonance imaging of the brain with spectroscopy was suggestive of Canavan disease, which was confirmed by chromatography of urinary organic acids.

Radiological clue to diagnosis of Canavan disease.

Canavan disease is an autosomal recessive leukodystrophy characterized by early onset developmental delay, initial hypotonia progressing to hypertonia, macrocephaly and blindness. The authors present an infant with these clinical features. MRI brain shows white matter changes with characteristic involvement of subcortical U fibres and MR spectroscopy shows the characteristic peak of N- acetyl aspartate. The importance of specific clinical features and imaging in the diagnosis of different leukodystrophies in resource and access limited settings is suggested.

A thematic review of scientific and family interests in Canavan Disease: where are the developmentalists?

BACKGROUND: Canavan Disease is a degenerative neurological condition resulting in a spongy deterioration of the brain. Much research has been conducted by the medical community regarding this condition, but little research can be found in the psychological literature. METHOD: A review of the scientific literature related to Canavan Disease using the Psychinfo and PubMed databases was conducted covering a 5-year span from 2006 through 2011. Concurrently, a review of parent initiated topics found on the most popular Canavan Disease Internet discussion board was conducted for comparison purposes. RESULTS: When comparing the topics discussed and information sought among parents with the themes noted in the extant scientific literature, researchers found an exceedingly small overlap between the two communities of interest. In the scientific literature, published research on Canavan Disease focused on three areas: the biochemistry of Canavan Disease, diagnosis and genetic counselling, and clinical therapeutic approaches in Canavan Disease. Of the 42 unique topics raised on a popular Internet discussion board, however, only three (7%) fell into the category of diagnosis and genetic counselling, none (0%) fell into the category of the biochemistry of Canavan Disease, and four fell into the category of clinical therapeutic approaches in Canavan Disease (10%). Of the four posts addressing clinical therapeutic approaches to Canavan Disease, only one post truly overlapped with the topics addressed by the scientific community. Worded differently, while these three categories comprise 100% of the extant scientific literature regarding Canavan Disease, they comprise only 17% of the parent-raised topics. The remaining 83% of parent-raised topics addressed concerns not currently being focusing upon by the scientific community, namely, non-medical practical issues, information regarding specific characteristics of Canavan Disease, non-medical developmental and quality of life issues, and day-to-day developmental and medical concerns. CONCLUSION: By comparing the extant literature on Canavan Disease with the topics of interest raised by parents and caregivers, it seems clear that there is a significant 'underlap' of topics raised by these two communities of interest, one that may reflect a lack of sensitivity on the part of the scientific community to meet the needs of this population of knowledge seekers. It is the suggestion of these authors that developmental psychology may be the appropriate scientific field within which to address this need and fill this gap in the current literature.

Molecular characterisation and prenatal diagnosis of Asparto-acylase deficiency (Canavan disease)--report of two novel and two known mutations from the Indian subcontinent.

OBJECTIVES: To establish a technique for mutation identification and prenatal screening in confirmed cases of Canavan disease. METHOD: Mutations in ASPA gene were identified by sequencing. Six exons of ASPA gene were amplified using intronic primers flanking the exons and then sequenced on ABI 3500Dx automated unit. This technique was used to identify mutations in three cases of Canavan disease. Prenatal diagnosis was performed in two families. RESULTS: Two reported mutations c.162 C > A (p.Asn54Lys) and c.859 G > A (p.Ala287Thr) were identified in two different cases of Canavan disease. Third case was compound heterozygous for two novel mutations (c.728 T > G, p.Ile243Ser; c.902 T > C, p.Leu301Pro). Prenatal diagnosis was performed in three pregnancies in two families, two affected fetuses and one unaffected fetus were identified. CONCLUSIONS: Molecular characterization of Canavan disease helps identify the cause at genetic level, thus confirming diagnosis and enabling identification of carriers in the family. Though enzyme assay and NAA measurement allows diagnosis and prenatal diagnosis of Canavan diasease, molecular methods have the advantage of bringing accuracy in prenatal testing with an earlier result. This is the first case report of mutation studies in Canavan disease from Indian subcontinent.

Lithium citrate as treatment of Canavan disease.

Canavan disease is a rare autosomal recessive leukodystrophy characterized by abnormal accumulation of N-acetyl aspartate (NAA) in brain white matter. Currently, there is no cure for this disease, and management of patients consists mainly of treating symptoms. We describe a 3-month-old girl who was hospitalized for poor head control and decreased muscle tone. A battery of laboratory and genetic (homozygous mutation p.C218X) analysis revealed the presence of Canavan disease. Lithium citrate was initiated at a dosage of 45 mg/kg per day after diagnosis. Periodic controls of thyroid and liver function, and lithium levels in blood showed that this drug was sure and well tolerated. After 1 year of treatment, NAA levels decreased by approximately 20% in the brain region, urinary NAA levels showed a reduction of 80%, and patient improved alertness and visual tracking but continued with no heat support, axial hypotonia, and spastic diplegia. In our patient, the results obtained after drug administration are important with respect to the decrease in NAA and more discreet in clinical improvement. However, given the absence of adverse effects and limited treatment options, lithium citrate may be a good alternative to stop the progression of the disease and improve the quality of life of patients.