Propionic acidemia (PA) is a severe autosomal recessive metabolic disease caused by deficiency of... more Propionic acidemia (PA) is a severe autosomal recessive metabolic disease caused by deficiency of propionyl-CoA carboxylase (PCC). We studied PA transgenic (Pat) mice that lack endogenous PCC but express a hypoactive human PCCA cDNA, permitting their survival. Pat cohorts followed from 3 to 20 weeks of age showed growth failure and lethal crises of lethargy and hyperammonemia, commoner in males (27/50, 54%) than in females (11/52, 21%) and occurring mainly in Pat mice with the most severe growth deficiency. Groups of Pat mice were studied under basal conditions (P-Ba mice) and during acute crises (P-Ac). Plasma acylcarnitines in P-Ba mice, compared to controls, showed markedly elevated C3- and low C2-carnitine, with a further decrease in C2-carnitine in P-Ac mice. These clinical and biochemical findings resemble those of human PA patients. Liver acyl-CoA measurements showed that propionyl-CoA was a minor species in controls (propionyl-CoA/acetyl-CoA ratio, 0.09). In contrast, in P-Ba liver the ratio was 1.4 and in P-Ac liver, 13, with concurrent reductions of the levels of acetyl-CoA and other acyl-CoAs. Plasma ammonia levels in control, P-Ba and P-Ac mice were 109 ± 10, 311 ± 48 and 551 ± 61 μmol/L respectively. Four-week administration to Pat mice, of carglumate (N-carbamyl-L-glutamic acid), an analogue of N-carbamylglutamate, the product of the only acyl-CoA-requiring reaction directly related to the urea cycle, was associated with increased food consumption, improved growth and absence of fatal crises. Pat mice showed many similarities to human PA patients and provide a useful model for studying tissue pathophysiology and treatment outcomes.
To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry... more To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration. Results: As of December 1, 2010, 130 sites in 45 countries have uploaded a total of 25,114 percentile data points, 565,232 analyte results of true positive cases with 64 conditions, and 5,341 cutoff values. The average rate of submission of true positive cases between December 1, 2008, and December 1, 2010, was 5.1 cases/day. This cumulative evidence generated 91 high and 23 low cutoff target ranges. The overall proportion of cutoff values within the respective See Acknowledgements for author affiliation.
Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with uniq... more Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct Arich PAMs and provide structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes. 14 glycemia and weight loss were normalized in the treatment groups (Fig. 4G,H). 3 Therefore, AAV8-mediated delivery of St1Cas9 in neonatal mice can result in efficient DNA cleavage, stable genetic modification, and phenotypic correction by rewiring a 3 metabolic pathway through gene inactivation. 3 To corroborate these findings we targeted Pck1, the gene encoding phosphoenolpyruvate 3 carboxykinase 1, which plays a broad role in integrating hepatic energy metabolism and 3 gluconeogenesis (Yang et al. 2009). Mice with a liver-specific deletion of the gene are 3 viable but display an impaired response to fasting (She et al. 2000). Neonatal 2 days old 3 C57BL/6N (wild-type) pups were injected with AAV8-St1Cas9 v3 targeting Pck1 and at 3 6 weeks of age, they were fasted for 24 hours and killed for metabolic profiling and 3 evaluating gene disruption efficacy (Supplemental Fig. S6D,E). Systemic delivery via a 3 single neonatal injection resulted in substantial hepatic Pck1 gene disruption 3 (Supplemental Fig. S6D). Plasma and hepatic triglyceride content were also markedly 3 increased (Supplemental Fig. S6E). However, we found no change in circulating free 3 fatty acid levels and hepatic glycogen stores were not depleted, suggesting that the 3 observed phenotype may be intermediary to the one described in a prenatal hepatic 3 knockout model (Supplemental Fig. S6E) (She et al. 2000). We speculate that normal 3 PCK1 in any non-targeted hepatocytes can partially compensate for the loss-of-function 3 resulting from in vivo editing. Nevertheless, AAV8-mediated delivery of St1Cas9 in 3 neonatal mice can efficiently disrupt the function of a key metabolic enzyme leading to 3 clear and substantial phenotype in vivo. Collectively, these data support the notion that 3 St1Cas9 can be engineered as a powerful tool for in vivo genome editing.
The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological inno... more The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) and define variables required for robust and efficient editing in vitro and in vivo. We show that diverse bacterial strains encode divergent St1Cas9s with altered protospacer adjacent motif (PAM) binding specificity offering a simple solution to expand targeting density. Expression of holo-St1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver efficiently rewires metabolic pathways due to robust DNA cleavage activity that is comparable to Staphylococcus aureus Cas9 (SaCas9) in vivo. Lastly, we identified potent anti-CRISPR proteins that differentially inhibit the activity of ...
A high level of succinylacetone (SA) in blood is a sensitive, specific marker for the screening a... more A high level of succinylacetone (SA) in blood is a sensitive, specific marker for the screening and diagnosis of hepatorenal tyrosinemia (HT1, MIM 276700). HT1 is caused by mutations in the FAH gene, resulting in deficiency of fumarylacetoacetate hydrolase. HT1 newborns are usually clinically asymptomatic, but have coagulation abnormalities revealing liver dysfunction. Treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of HT1. Two newborns screened positive for SA but had normal coagulation testing. Plasma and urine SA levels were 3-5 fold above the reference range but were markedly lower than in typical HT1. Neither individual received nitisinone or dietary therapy. They remain clinically normal, currently aged 9 and 15 years. Each was a compound heterozygote, having a splicing variant in trans with a prevalent "pseudodeficient" FAH allele, c.1021C > T (p.Arg341Trp), which confers partial FAH activity. All...
A high level of succinylacetone (SA) in blood is a sensitive, specific newborn screening marker f... more A high level of succinylacetone (SA) in blood is a sensitive, specific newborn screening marker for hepatorenal tyrosinemia type 1 (HT1, MIM 276700) caused by deficiency of fumarylacetoacetate hydrolase (FAH). Newborns with HT1 are usually clinically asymptomatic but show liver dysfunction with coagulation abnormalities (prolonged prothrombin time and/or high international normalised ratio). Early treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of severe liver disease and neurological crises. Six newborns referred for hypersuccinylacetonaemia but who had normal coagulation testing on initial evaluation had sequence variants in the GSTZ1 gene, encoding maleylacetoacetate isomerase (MAAI), the enzyme preceding FAH in tyrosine degradation. Initial plasma SA levels ranged from 233 to 1282 nmol/L, greater than normal (<24 nmol/L) but less than the initial values of patients with HT1 (16 944-74 377 nmol/L, n=15). Four i...
Insulin increases plasma nonesterified fatty acid (NEFA) clearance in humans, but whether this is... more Insulin increases plasma nonesterified fatty acid (NEFA) clearance in humans, but whether this is independent of change in plasma NEFA appearance is currently unknown. Nine nondiabetic men (age: 28 ± 3 yr, body mass index: 27.2 ± 1.7 kg/m2) underwent euglycemic clamps to maintain low (LINS) vs. high (HINS) physiological insulin levels for 6 h. An intravenous infusion of heparin + Intralipid (HI) was performed during 4 of the 6 h of the clamps (in the last 4 h at LINS and in the first 4 h at HINS), whereas saline infusion (SAL) was administered in the remaining 2 h to modulate plasma NEFA levels independently of plasma insulin levels. Four experimental conditions were obtained in each individual: LINS with saline (LINS/SAL) and with HI infusion (LINS/HI) and HINS with saline (HINS/SAL) and with HI infusion (HINS/HI). Plasma palmitate appearance during HINS/SAL was lower than during the three other experimental conditions ( P &lt; 0.05). In contrast, plasma linoleate appearance, as expected, was increased by HI independently of insulin level ( P &lt; 0.02). Plasma palmitate clearance during HINS/SAL was higher than LINS/SAL and LINS/HI ( P &lt; 0.008), and this increase was blunted during HINS/HI. We observed a linear decrease in plasma palmitate clearance with increasing plasma NEFA appearance independent of insulin levels. Plasma NEFA levels increased exponentially with increase in plasma NEFA appearance. We conclude that insulin stimulates plasma NEFA clearance by reducing the endogenous appearance rate of NEFA. The relationship between plasma NEFA level and appearance rate is nonlinear.
Fabry disease is a complex, multisystemic and clinically heterogeneous disease, in which the urin... more Fabry disease is a complex, multisystemic and clinically heterogeneous disease, in which the urinary excretion of globotriaosylceramide (Gb 3), the principal substrate of the deficient enzyme, a-galactosidase A, is more prominent than the increased concentrations of the lipid in the plasma of affected hemizygotes and heterozygotes. We have developed and validated a simultaneous analysis of Gb 3 and creatinine in a 2.6-min run using filter paper discs saturated with urine and analyzed by LC-MS/MS. Using this method, we studied the relationship between urinary levels of total Gb 3 /creatinine excretion and four types of mutations in the GLA gene (missense, nonsense, frameshift, and splice-site defects) in 32 children and 78 adult patients with Fabry disease. Forty-one patients were treated by enzyme replacement therapy and 69 were untreated. Our results show that the mean recoveries of Gb 3 and creatinine from the urine filter paper standards were 91% and 97%, respectively, with precision, reproducibility, and linearity within acceptable ranges. Statistical analysis using the independent variables of sex, age, types of mutations and treatment showed that the mutation factor has a statistically significant impact on urinary Gb 3 excretion (p = 0.0007). This means that the levels of urinary excretion of Gb 3 /creatinine in children and adults with Fabry disease are directly related to the types of mutations. The same correlation was found for the sex (p < 0.0001) and treatment (p = 0.0011). In conclusion, we studied 35 mutations in 110 children and adults with Fabry disease and found a significant correlation between the types of mutations and total Gb 3 excretion in Fabry patients.
on mobility. Pulmonary function vital capacity increased from 0.72 prior to treatment to 1.5 afte... more on mobility. Pulmonary function vital capacity increased from 0.72 prior to treatment to 1.5 after two years of enzyme therapy. The patient has experienced no adverse events since beginning enzyme replacement therapy. Infantile-onset Pompe disease is a progressive and eventually lethal muscle disorder. Enzyme replacement therapy in our patient has both stopped and reversed the muscle disorder as witnessed by improvements in pulmonary function testing, muscle stamina studies, and observed endurance.
The Quebec Mass Urinary Screening Programme, initiated in 1971, has resulted in the screening of ... more The 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.
Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting... more Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting from a deficiency of the enzyme alpha-galactosidase A, and leading to the progressive accumulation of one biomarker, globotriaosylceramide (Gb(3)), predominantly elevated in the urine of these patients. We have developed a technique for the analysis of total Gb(3) in urine samples collected on filter paper, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a triple quadrupole instrument. Existing Gb(3) 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 Gb(3) 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 Gb(3) 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 Gb(3) 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.
The Journal of Clinical Endocrinology & Metabolism, 2009
Context: Increased plasma nonesterified fatty acid (NEFA) appearance during enhanced intravascula... more Context: Increased plasma nonesterified fatty acid (NEFA) appearance during enhanced intravascular triacylglycerol (TG) lipolysis is a marker of metabolic adipose tissue dysfunction and may lead to the development of insulin resistance. The relationship between total and high molecular weight (HMW) adiponectin levels, NEFA appearance, and total TG lipolytic capacity has not been previously studied in humans. Objectives: Our objective was to determine whether total and HMW adiponectin plasma levels are associated with plasma NEFA level and appearance, and with total TG lipolytic rate during enhanced intravascular TG lipolysis in men. Design: This was a cross-sectional metabolic study. Setting: The study was performed at an academic clinical research center. Participants: There were 15 healthy men (mean ± sd body mass index 25.5 ± 4.7 kg/m2) aged 21–50 yr (mean ± sd 31.1 ± 10.2) without first-degree relatives with type 2 diabetes included in the study. Interventions: Pancreatic clamps...
Propionic acidemia (PA) is a severe autosomal recessive metabolic disease caused by deficiency of... more Propionic acidemia (PA) is a severe autosomal recessive metabolic disease caused by deficiency of propionyl-CoA carboxylase (PCC). We studied PA transgenic (Pat) mice that lack endogenous PCC but express a hypoactive human PCCA cDNA, permitting their survival. Pat cohorts followed from 3 to 20 weeks of age showed growth failure and lethal crises of lethargy and hyperammonemia, commoner in males (27/50, 54%) than in females (11/52, 21%) and occurring mainly in Pat mice with the most severe growth deficiency. Groups of Pat mice were studied under basal conditions (P-Ba mice) and during acute crises (P-Ac). Plasma acylcarnitines in P-Ba mice, compared to controls, showed markedly elevated C3- and low C2-carnitine, with a further decrease in C2-carnitine in P-Ac mice. These clinical and biochemical findings resemble those of human PA patients. Liver acyl-CoA measurements showed that propionyl-CoA was a minor species in controls (propionyl-CoA/acetyl-CoA ratio, 0.09). In contrast, in P-Ba liver the ratio was 1.4 and in P-Ac liver, 13, with concurrent reductions of the levels of acetyl-CoA and other acyl-CoAs. Plasma ammonia levels in control, P-Ba and P-Ac mice were 109 ± 10, 311 ± 48 and 551 ± 61 μmol/L respectively. Four-week administration to Pat mice, of carglumate (N-carbamyl-L-glutamic acid), an analogue of N-carbamylglutamate, the product of the only acyl-CoA-requiring reaction directly related to the urea cycle, was associated with increased food consumption, improved growth and absence of fatal crises. Pat mice showed many similarities to human PA patients and provide a useful model for studying tissue pathophysiology and treatment outcomes.
To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry... more To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration. Results: As of December 1, 2010, 130 sites in 45 countries have uploaded a total of 25,114 percentile data points, 565,232 analyte results of true positive cases with 64 conditions, and 5,341 cutoff values. The average rate of submission of true positive cases between December 1, 2008, and December 1, 2010, was 5.1 cases/day. This cumulative evidence generated 91 high and 23 low cutoff target ranges. The overall proportion of cutoff values within the respective See Acknowledgements for author affiliation.
Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with uniq... more Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct Arich PAMs and provide structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes. 14 glycemia and weight loss were normalized in the treatment groups (Fig. 4G,H). 3 Therefore, AAV8-mediated delivery of St1Cas9 in neonatal mice can result in efficient DNA cleavage, stable genetic modification, and phenotypic correction by rewiring a 3 metabolic pathway through gene inactivation. 3 To corroborate these findings we targeted Pck1, the gene encoding phosphoenolpyruvate 3 carboxykinase 1, which plays a broad role in integrating hepatic energy metabolism and 3 gluconeogenesis (Yang et al. 2009). Mice with a liver-specific deletion of the gene are 3 viable but display an impaired response to fasting (She et al. 2000). Neonatal 2 days old 3 C57BL/6N (wild-type) pups were injected with AAV8-St1Cas9 v3 targeting Pck1 and at 3 6 weeks of age, they were fasted for 24 hours and killed for metabolic profiling and 3 evaluating gene disruption efficacy (Supplemental Fig. S6D,E). Systemic delivery via a 3 single neonatal injection resulted in substantial hepatic Pck1 gene disruption 3 (Supplemental Fig. S6D). Plasma and hepatic triglyceride content were also markedly 3 increased (Supplemental Fig. S6E). However, we found no change in circulating free 3 fatty acid levels and hepatic glycogen stores were not depleted, suggesting that the 3 observed phenotype may be intermediary to the one described in a prenatal hepatic 3 knockout model (Supplemental Fig. S6E) (She et al. 2000). We speculate that normal 3 PCK1 in any non-targeted hepatocytes can partially compensate for the loss-of-function 3 resulting from in vivo editing. Nevertheless, AAV8-mediated delivery of St1Cas9 in 3 neonatal mice can efficiently disrupt the function of a key metabolic enzyme leading to 3 clear and substantial phenotype in vivo. Collectively, these data support the notion that 3 St1Cas9 can be engineered as a powerful tool for in vivo genome editing.
The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological inno... more The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) and define variables required for robust and efficient editing in vitro and in vivo. We show that diverse bacterial strains encode divergent St1Cas9s with altered protospacer adjacent motif (PAM) binding specificity offering a simple solution to expand targeting density. Expression of holo-St1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver efficiently rewires metabolic pathways due to robust DNA cleavage activity that is comparable to Staphylococcus aureus Cas9 (SaCas9) in vivo. Lastly, we identified potent anti-CRISPR proteins that differentially inhibit the activity of ...
A high level of succinylacetone (SA) in blood is a sensitive, specific marker for the screening a... more A high level of succinylacetone (SA) in blood is a sensitive, specific marker for the screening and diagnosis of hepatorenal tyrosinemia (HT1, MIM 276700). HT1 is caused by mutations in the FAH gene, resulting in deficiency of fumarylacetoacetate hydrolase. HT1 newborns are usually clinically asymptomatic, but have coagulation abnormalities revealing liver dysfunction. Treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of HT1. Two newborns screened positive for SA but had normal coagulation testing. Plasma and urine SA levels were 3-5 fold above the reference range but were markedly lower than in typical HT1. Neither individual received nitisinone or dietary therapy. They remain clinically normal, currently aged 9 and 15 years. Each was a compound heterozygote, having a splicing variant in trans with a prevalent "pseudodeficient" FAH allele, c.1021C > T (p.Arg341Trp), which confers partial FAH activity. All...
A high level of succinylacetone (SA) in blood is a sensitive, specific newborn screening marker f... more A high level of succinylacetone (SA) in blood is a sensitive, specific newborn screening marker for hepatorenal tyrosinemia type 1 (HT1, MIM 276700) caused by deficiency of fumarylacetoacetate hydrolase (FAH). Newborns with HT1 are usually clinically asymptomatic but show liver dysfunction with coagulation abnormalities (prolonged prothrombin time and/or high international normalised ratio). Early treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of severe liver disease and neurological crises. Six newborns referred for hypersuccinylacetonaemia but who had normal coagulation testing on initial evaluation had sequence variants in the GSTZ1 gene, encoding maleylacetoacetate isomerase (MAAI), the enzyme preceding FAH in tyrosine degradation. Initial plasma SA levels ranged from 233 to 1282 nmol/L, greater than normal (<24 nmol/L) but less than the initial values of patients with HT1 (16 944-74 377 nmol/L, n=15). Four i...
Insulin increases plasma nonesterified fatty acid (NEFA) clearance in humans, but whether this is... more Insulin increases plasma nonesterified fatty acid (NEFA) clearance in humans, but whether this is independent of change in plasma NEFA appearance is currently unknown. Nine nondiabetic men (age: 28 ± 3 yr, body mass index: 27.2 ± 1.7 kg/m2) underwent euglycemic clamps to maintain low (LINS) vs. high (HINS) physiological insulin levels for 6 h. An intravenous infusion of heparin + Intralipid (HI) was performed during 4 of the 6 h of the clamps (in the last 4 h at LINS and in the first 4 h at HINS), whereas saline infusion (SAL) was administered in the remaining 2 h to modulate plasma NEFA levels independently of plasma insulin levels. Four experimental conditions were obtained in each individual: LINS with saline (LINS/SAL) and with HI infusion (LINS/HI) and HINS with saline (HINS/SAL) and with HI infusion (HINS/HI). Plasma palmitate appearance during HINS/SAL was lower than during the three other experimental conditions ( P &lt; 0.05). In contrast, plasma linoleate appearance, as expected, was increased by HI independently of insulin level ( P &lt; 0.02). Plasma palmitate clearance during HINS/SAL was higher than LINS/SAL and LINS/HI ( P &lt; 0.008), and this increase was blunted during HINS/HI. We observed a linear decrease in plasma palmitate clearance with increasing plasma NEFA appearance independent of insulin levels. Plasma NEFA levels increased exponentially with increase in plasma NEFA appearance. We conclude that insulin stimulates plasma NEFA clearance by reducing the endogenous appearance rate of NEFA. The relationship between plasma NEFA level and appearance rate is nonlinear.
Fabry disease is a complex, multisystemic and clinically heterogeneous disease, in which the urin... more Fabry disease is a complex, multisystemic and clinically heterogeneous disease, in which the urinary excretion of globotriaosylceramide (Gb 3), the principal substrate of the deficient enzyme, a-galactosidase A, is more prominent than the increased concentrations of the lipid in the plasma of affected hemizygotes and heterozygotes. We have developed and validated a simultaneous analysis of Gb 3 and creatinine in a 2.6-min run using filter paper discs saturated with urine and analyzed by LC-MS/MS. Using this method, we studied the relationship between urinary levels of total Gb 3 /creatinine excretion and four types of mutations in the GLA gene (missense, nonsense, frameshift, and splice-site defects) in 32 children and 78 adult patients with Fabry disease. Forty-one patients were treated by enzyme replacement therapy and 69 were untreated. Our results show that the mean recoveries of Gb 3 and creatinine from the urine filter paper standards were 91% and 97%, respectively, with precision, reproducibility, and linearity within acceptable ranges. Statistical analysis using the independent variables of sex, age, types of mutations and treatment showed that the mutation factor has a statistically significant impact on urinary Gb 3 excretion (p = 0.0007). This means that the levels of urinary excretion of Gb 3 /creatinine in children and adults with Fabry disease are directly related to the types of mutations. The same correlation was found for the sex (p < 0.0001) and treatment (p = 0.0011). In conclusion, we studied 35 mutations in 110 children and adults with Fabry disease and found a significant correlation between the types of mutations and total Gb 3 excretion in Fabry patients.
on mobility. Pulmonary function vital capacity increased from 0.72 prior to treatment to 1.5 afte... more on mobility. Pulmonary function vital capacity increased from 0.72 prior to treatment to 1.5 after two years of enzyme therapy. The patient has experienced no adverse events since beginning enzyme replacement therapy. Infantile-onset Pompe disease is a progressive and eventually lethal muscle disorder. Enzyme replacement therapy in our patient has both stopped and reversed the muscle disorder as witnessed by improvements in pulmonary function testing, muscle stamina studies, and observed endurance.
The Quebec Mass Urinary Screening Programme, initiated in 1971, has resulted in the screening of ... more The 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.
Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting... more Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting from a deficiency of the enzyme alpha-galactosidase A, and leading to the progressive accumulation of one biomarker, globotriaosylceramide (Gb(3)), predominantly elevated in the urine of these patients. We have developed a technique for the analysis of total Gb(3) in urine samples collected on filter paper, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a triple quadrupole instrument. Existing Gb(3) 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 Gb(3) 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 Gb(3) 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 Gb(3) 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.
The Journal of Clinical Endocrinology & Metabolism, 2009
Context: Increased plasma nonesterified fatty acid (NEFA) appearance during enhanced intravascula... more Context: Increased plasma nonesterified fatty acid (NEFA) appearance during enhanced intravascular triacylglycerol (TG) lipolysis is a marker of metabolic adipose tissue dysfunction and may lead to the development of insulin resistance. The relationship between total and high molecular weight (HMW) adiponectin levels, NEFA appearance, and total TG lipolytic capacity has not been previously studied in humans. Objectives: Our objective was to determine whether total and HMW adiponectin plasma levels are associated with plasma NEFA level and appearance, and with total TG lipolytic rate during enhanced intravascular TG lipolysis in men. Design: This was a cross-sectional metabolic study. Setting: The study was performed at an academic clinical research center. Participants: There were 15 healthy men (mean ± sd body mass index 25.5 ± 4.7 kg/m2) aged 21–50 yr (mean ± sd 31.1 ± 10.2) without first-degree relatives with type 2 diabetes included in the study. Interventions: Pancreatic clamps...
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Papers by Denis Cyr