Christiane Auray-Blais

Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria

Solute carrier family 1, member 1 (SLC1A1; also known as EAAT3 and EAAC1) is the major epithelial transporter of glutamate and aspartate in the kidneys and intestines of rodents. Within the brain, SLC1A1 serves as the predominant neuronal glutamate transporter and buffers the synaptic release of the excitatory neurotransmitter glutamate within the interneuronal synaptic cleft. Recent studies have also revealed that polymorphisms in SLC1A1 are associated with obsessive-compulsive disorder (OCD) in early-onset patient cohorts. Here we report that SLC1A1 mutations leading to substitution of arginine to tryptophan at position 445 (R445W) and deletion of isoleucine at position 395 (I395del) cause human dicarboxylic aminoaciduria, an autosomal recessive disorder of urinary glutamate and aspartate transport that can be associated with mental retardation. These mutations of conserved residues impeded or abrogated glutamate and cysteine transport by SLC1A1 and led to near-absent surface expression in a canine kidney cell line. These findings provide evidence that SLC1A1 is the major renal transporter of glutamate and aspartate in humans and implicate SLC1A1 in the pathogenesis of some neurological disorders.

Efficient analysis of urinary glycosaminoglycans by LC-MS/MS in mucopolysaccharidoses type I, II and VI

Mucopolysaccharidoses (MPSs) are complex storage disorders caused by specific lysosomal enzyme deficiencies, resulting in the accumulation of glycosaminoglycans (GAGs) in urine, plasma, as well as in various tissues. We devised and validated a straightforward, but accurate and precise tandem mass spectrometry methodology coupled to high performance liquid chromatography (LC-MS/MS) for the quantification of GAGs in urine. The method is applicable to the investigation of patients with MPS I, II, and VI, by quantifying dermatan sulfate (DS) and heparan sulfate (HS) in urine. We analyzed urine samples from 28 MPS patients, aged 1 to 42 years, and 55 control subjects (41 days to 18 years old). Levels of DS and HS in urine from healthy controls of all ages were below the limit of quantification. The levels of DS and HS in urine from 6 treated patients with MPS I were lower than in 6 untreated patients in DS (0.7-45 vs 9.3-177 mg/mmol creat) and HS (0-123 mg/mmol creatinine vs 38-418 mg/mmol creatinine); similar results were obtained for 9 patients with MPS II and 7 patients with MPS VI. Analyses were performed on as little as 250 μL of urine. Methanolysis took 75 min per sample; the total analysis run time for each LC-MS/MS injection was 8 min. Results indicate that the method is applicable to a wide variety of situations in which high accuracy and precision are required, including the evaluation of the effectiveness of existing and emerging treatments.

How well does urinary lyso-Gb3 function as a biomarker in Fabry disease?

BACKGROUND Fabry disease is characterized by accumulation of glycosphingolipids, such as globotriaosylceramide (Gb(3)), in many tissues and body fluids. A novel plasma biomarker, globotriaosylsphingosine (lyso-Gb(3)), is increased in patients with the disease. Until now, lyso-Gb(3) was not detectable in urine, possibly because of the presence of interfering compounds. METHODS We undertook to: 1) characterize lyso-Gb(3) in urine; 2) develop a method to quantitate urinary lyso-Gb(3) by mass spectrometry; 3) evaluate urinary lyso-Gb(3) as a potential biomarker for Fabry disease; and 4) determine whether lyso-Gb(3) is an inhibitor of α-galactosidase A activity. We analyzed urinary lyso-Gb(3) from 83 Fabry patients and 77 healthy age-matched controls. RESULTS The intraday and interday bias and precision of the method were <15%. Increases in lyso-Gb(3)/creatinine correlated with the concentrations of Gb(3) (r(2)=0.43), type of mutations (p=0.0006), gender (p<0.0001) and enzyme replacement therapy status (p=0.0012). Urine from healthy controls contained no detectable lyso-Gb(3). Lyso-Gb(3) did not inhibit GLA activity in dried blood spots. Increased urinary excretion of lyso-Gb(3) of Fabry patients correlated well with a number of indicators of disease severity. CONCLUSION Lyso-Gb(3) is a reliable independent biomarker for clinically important characteristics of Fabry disease.

Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters

Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes.

Outcome of individuals with low-moderate methylmalonic aciduria detected through a neonatal screening program

BACKGROUND The clinical spectrum of methylmalonic aciduria (MMAuria) ranges from severe, neonatal acidosis to benign asymptomatic organic aciduria. In 1975, screening for MMAuria was established in the province of Quebec. Although newborn screening programs facilitate presymptomatic detection and treatment and also detect asymptomatic variants, uncertainties about potential long-term hazards of mild to moderate elevations of MMA create concern. The objective of this study was to examine the outcome of individuals excreting low to intermediate quantities of MMA, ascertained by a newborn screening program. RESULTS AND STUDY DESIGN One hundred and thirty-six individuals with elevations of urinary MMA were initially identified by the screening program; 122 individuals were noted to have excretion of urinary MMA <1400 micromol/mmol creatinine. At follow-up assessment at 1 year of age, in 65 of these 122 individuals, the MMA excretion had resolved. Of the remaining individuals, 9 were lost to follow-up, 13 had symptoms, and the remaining 35 were free of symptoms. Among the 35 individuals with asymptomatic persistent MMAuria, MMA excretion has resolved in 13 over 1 year; 22 individuals exhibit persistent low-moderate MMAuria (range, 210 to 1133 micromol/mmol creatinine). CONCLUSION Follow-up examination of individuals in the latter asymptomatic cohort with persistent low-moderate MMAuria indicates normal somatic and cognitive outcomes.

Urinary Globotriaosylsphingosine-Related Biomarkers for Fabry Disease Targeted by Metabolomics

Fabry disease is a lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in glycosphingolipid accumulation in organs and tissues, including plasma and urine. Two disease-specific Fabry biomarkers have been identified and quantified in plasma and urine: globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)). The search continues for biomarkers that might be reliable indicators of disease severity and response to treatment. The main objective of this study was to target other urinary biomarkers using a time-of-flight mass spectrometry metabolomic approach. Urinary metabolites of 63 untreated Fabry patients and 59 controls were analyzed. A multivariate statistical analysis performed on a subset of male samples revealed seven novel Fabry biomarkers in urine, all lyso-Gb(3) analogues having modified sphingosine moieties. The empirical formulas of the sphingosine modifications were determined by exact mass measurements (- C(2)H(4), - C(2)H(4) + O, - H(2), - H(2) + O, + O, + H(2)O(2), + H(2)O(3)). We evaluated the relative concentration of lyso-Gb(3) and its seven analogues by measuring area counts for each analogue in all Fabry patients. All samples were normalized to creatinine. We found higher concentrations for males with Fabry disease compared to females. None of these biomarkers were detected in controls. To our knowledge, this is the first time that lyso-Gb(3)-related Fabry disease biomarkers are detected in urine.

Newborn Urine Screening Experience with over one Million Infants in the Quebec Network of Genetic Medicine

SummaryWe screened urine for chemical individuality in over 1 million newborn infants, by various chromatographic (thin-layer), chemical and spectrophotometric methods, 12 procedures in all. The programme is part of the Quebec Network of Genetic Medicine. Voluntary urine screening began in 1971 and has evolved with changes in choice of tests and times of sample collection. Urine samples were collected on filter paper at either 5, 14 or 21 days after birth; results were best with the 21-day test. Compliance is over 94% with the latter and over 98% with requests for repeat samples. Screening is centralized in one laboratory; follow-up diagnosis, counselling and management are done at four regional centres. Incidence of phenotypes ranged from 1:4300 live births (for expressed cystinuria alleles) to 1 per million (for hyperargininaemia). Over 20 inherited Mendelian disorders were identified. 30 patients required aggressive medical management. We show how this programme can be used for neuroblastoma screening.

Quebec neonatal mass urinary screening programme: From micromolecules to macromolecules

SummaryThe Quebec Mass Urinary Screening Programme, initiated in 1971, has resulted in the screening of more than 2 500 000 newborns in the province of Quebec for 25 inherited Mendelian disorders divided into two groups. The first group concerns urea cycle disorders (citrullinaemia, hyperargininaemia, argininosuccinic aciduria), ketotic hyperglycinaemia, and organic acidurias (methylmalonic aciduria, glutaric aciduria type I, etc.); the second group relates to disorders of amino acid metabolism (cystathioninuria, prolidase deficiency, etc.) and transport (Fanconi syndrome, cystinurias, Hartnup syndrome, etc.). The main goal of the Programme is to detect and prevent these genetic diseases, some detectable only in urine, before the onset of clinical symptoms. A multiplex thin-layer chromatography methodology was developed, in which metabolites in urine are resolved and visualized by the sequential application of four different reagents to detect aminoacidopathies and organic acidurias. The technique is simple, reproducible, inexpensive and rapid, allowing the analysis of 500 samples daily by a single technician. The voluntary compliance of the parents is excellent, averaging 90% per year. Over the years, we have established a dynamic process, developing techniques or new reagents to detect as many treatable disorders as possible, now evaluating macromolecules associated with lysosomal storage disorders, mainly globotriaosylceramide (Gb3) for Fabry disease. We present here the methodology, infrastructure in place, results and recent statistics of the well-established Quebec Mass Urinary Screening Programme. We also report a study by tandem mass spectrometric analysis of urinary Gb3 in Fabry disease for the follow-up and monitoring of Fabry patients, as well as for its possible application to mass and high-risk screening programmes.

Development of a filter paper method potentially applicable to mass and high-risk urinary screenings for Fabry disease

SummaryFabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting from a deficiency of the enzyme α-galactosidase A, and leading to the progressive accumulation of one biomarker, globotriaosylceramide (Gb3), predominantly elevated in the urine of these patients. We have developed a technique for the analysis of total Gb3 in urine samples collected on filter paper, using liquid chromatography–tandem mass spectrometry (LC-MS/MS) with a triple quadrupole instrument. Existing Gb3 techniques being both time- and labour-intensive, this filter paper method eliminates lipid extraction, glycolipid isolation, centrifugation and evaporation steps, while maintaining sensitivity and efficiency. The stability of Gb3 on filter paper was good for a 7-week period under different temperature conditions. Normal control values were established and the technique was tested with anonymized samples from Fabry hemizygotes and heterozygotes. The levels of total Gb3 in all classical hemizygotes were well above the control values and all heterozygotes, except two nonexcretors, were above the reference level. The proposed novel filter paper method favours the collection, storage and shipment of samples. It is simple and efficient for a feasibility study, potentially applicable to the determination of total urinary Gb3 in the newborn population as part of a screening programme, and could also be used in high-risk screening laboratories. Since the incidence of Fabry disease is hard to establish, owing to the heterogeneous clinical expression of the visible phenotype, this feasibility study could help determine its actual incidence in the Quebec population.

Multiplex Analysis of Novel Urinary Lyso-Gb3-Related Biomarkers for Fabry Disease by Tandem Mass Spectrometry

Fabry disease is a lysosomal storage disorder caused by the absence or reduction of α-galactosidase A enzyme activity. The enzymatic deficiency results in the impaired catabolism of neutral sphingolipids with terminal α-galactosyl residues and subsequent accumulation in several tissues. Biomarkers reflecting disease severity and progression, the response to therapeutic intervention, and details of molecular pathogenesis are needed. Until now, two sphingolipids were targeted as biomarkers in urine and plasma of Fabry patients: globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)). Using metabolomic approaches, our group recently discovered seven novel urinary lyso-Gb(3)-related Fabry disease biomarkers with mass-to-charge ratios (m/z) of 758, 774, 784, 800, 802, 820, and 836. All these biomarkers exhibited modifications of the lyso-Gb(3) sphingosine moiety. The aims of the present study were to devise and validate a specific tandem mass spectrometry multiplex methodology for the relative quantification of these seven analogues and to evaluate their urinary excretion levels in samples from 164 Fabry patients and 94 healthy controls. We found no detectable analogues in healthy controls, except for trace amounts of the analogue with m/z 836. Significant correlations were established between lyso-Gb(3) analogue levels in urine and gender (p < 0.001). Fabry males had higher excretion levels compared to females with the disease. Lyso-Gb(3) analogue levels correlated well with enzyme replacement therapy (ERT) status in males (p < 0.05). The urinary analogue distributions varied among Fabry patients. However, the analogues with m/z 802, 820, and 836 were generally more abundant in the majority of patients. Lyso-Gb(3) analogues are promising urinary biomarkers for Fabry disease.