Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.

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 A-rich PAMs and provide a 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.

The multiple facets of acetyl-CoA metabolism: Energetics, biosynthesis, regulation, acylation and inborn errors.

Acetyl-coenzyme A (Ac-CoA) is a core metabolite with essential roles throughout cell physiology. These functions can be classified into energetics, biosynthesis, regulation and acetylation of large and small molecules. Ac-CoA is essential for oxidative metabolism of glucose, fatty acids, most amino acids, ethanol, and of free acetate generated by endogenous metabolism or by gut bacteria. Ac-CoA cannot cross lipid bilayers, but acetyl groups from Ac-CoA can shuttle across membranes as part of carrier molecules like citrate or acetylcarnitine, or as free acetate or ketone bodies. Ac-CoA is the basic unit of lipid biosynthesis, providing essentially all of the carbon for the synthesis of fatty acids and of isoprenoid-derived compounds including cholesterol, coenzyme Q and dolichols. High levels of Ac-CoA in hepatocytes stimulate lipid biosynthesis, ketone body production and the diversion of pyruvate metabolism towards gluconeogenesis and away from oxidation; low levels exert opposite effects. Acetylation changes the properties of molecules. Acetylation is necessary for the synthesis of acetylcholine, acetylglutamate, acetylaspartate and N-acetyl amino sugars, and to metabolize/eliminate some xenobiotics. Acetylation is a major post-translational modification of proteins. Different types of protein acetylation occur. The most-studied form occurs at the epsilon nitrogen of lysine residues. In histones, lysine acetylation can alter gene transcription. Acetylation of other proteins has diverse, often incompletely-documented effects. Inborn errors related to Ac-CoA feature a broad spectrum of metabolic, neurological and other features. To date, a small number of studies of animals with inborn errors of CoA thioesters has included direct measurement of acyl-CoAs. These studies have shown that low levels of tissue Ac-CoA correlate with the development of clinical signs, hinting that shortage of Ac-CoA may be a recurrent theme in these conditions. Low levels of Ac-CoA could potentially disrupt any of its roles.

Propionic acidemia in mice: Liver acyl-CoA levels and clinical course.

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.

Hypersuccinylacetonaemia and normal liver function in maleylacetoacetate isomerase deficiency.

BACKGROUND: 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. METHODS AND RESULTS: 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 individuals were homozygous for c.449C>T (p.Ala150Val). One was compound heterozygous for c.259C>T (p.Arg87Ter) and an intronic sequence variant. In one, a single heterozygous GSTZ1 sequence variant was identified, c.295G>A (p.Val99Met). Bacterial expression of p.Ala150Val and p.Val99Met revealed low MAAI activity. The six individuals with mild hypersuccinylacetonaemia (MHSA) were not treated with diet or nitisinone. Their clinical course has been normal for up to 13 years. CONCLUSIONS: MHSA can be caused by sequence variants in GSTZ1. Such individuals have thus far remained asymptomatic despite receiving no specific treatment.

HSD10 mitochondrial disease: p.Leu122Val variant, mild clinical phenotype, and founder effect in French-Canadian patients from Quebec.

BACKGROUND: HSD10 mitochondrial disease (HSD10MD), originally described as a deficiency of 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD), is a rare X-linked disorder of a moonlighting protein encoded by the HSD17B10. The diagnosis is usually first suspected on finding elevated isoleucine degradation metabolites in urine, reflecting decreased MHBD activity. However, it is now known that clinical disease pathogenesis reflects other independent functions of the HSD10 protein; particularly its essential role in mitochondrial transcript processing and tRNA maturation. The classical phenotype of HSD10MD in affected males is an infantile-onset progressive neurodegenerative disorder associated with severe mitochondrial dysfunction. PATIENTS, METHODS, AND RESULTS: In four unrelated families, we identified index patients with MHBD deficiency, which implied a diagnosis of HSD10MD. Each index patient was independently investigated because of neurological or developmental concerns. All had persistent elevations of urinary 2-methyl-3-hydroxybutyric acid and tiglylglycine. Analysis of HSD17B10 identified a single missense variant, c.364C>G, p.Leu122Val, in each case. This rare variant (1/183336 alleles in gnomAD) was previously reported in one Dutch patient and was described as pathogenic. The geographic origins of our families and results of haplotype analysis together provide evidence of a founder effect for this variant in Quebec. Notably, we identified an asymptomatic hemizygous adult male in one family, while a second independent genetic disorder contributed substantially to the clinical phenotypes observed in probands from two other families. CONCLUSION: The phenotype associated with p.Leu122Val in HSD17B10 currently appears to be attenuated and nonprogressive. This report widens the spectrum of phenotypic severity of HSD10MD and contributes to genotype-phenotype correlation. At present, we consider p.Leu122Val a "variant of uncertain significance." Nonetheless, careful follow-up of our patients remains advisable, to assess long-term clinical course and ensure appropriate management. It will also be important to identify other potential patients in our population and to characterize their phenotype.

Urinary globotriaosylceramide excretion correlates with the genotype in children and adults with Fabry disease.

Fabry disease is a complex, multisystemic and clinically heterogeneous disease, in which the urinary excretion of globotriaosylceramide (Gb3), the principal substrate of the deficient enzyme, alpha-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 Gb3 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 Gb3/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 Gb3 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 Gb3 excretion (p = 0.0007). This means that the levels of urinary excretion of Gb3/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 Gb3 excretion in Fabry patients.

Mildly elevated succinylacetone and normal liver function in compound heterozygotes with pathogenic and pseudodeficient FAH alleles.

BACKGROUND: 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. OBSERVATIONS: 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 newborns identified with mild hypersuccinylacetonemia in Québec have had genetic deficiencies of tyrosine degradation: either deficiency of the enzyme preceding FAH, maleylacetoacetate isomerase, or partial deficiency of FAH itself. CONCLUSION: Compound heterozygotes for c.1021C > T (p.Arg341Trp) and a severely deficient FAH allele have mild hypersuccinylacetonemia and to date they have remained asymptomatic without treatment. It is important to determine the long term outcome of such individuals.

A GC/MS validated method for the nanomolar range determination of succinylacetone in amniotic fluid and plasma: an analytical tool for tyrosinemia type I.

A sensitive and accurate stable isotope dilution GC/MS assay was developed and validated for the quantification of succinylacetone (SA) in plasma and amniotic fluid (AF). SA is pathognonomic for tyrosinemia type I, a genetic disorder caused by a reduced activity of fumarylacetoacetate hydrolase (FAH). In untreated patients, SA can easily be measured in plasma and urine because the expected concentrations are in the micromol/L range. Due to a founder effect, the province of Quebec has an unusually high prevalence of tyrosinemia type I, hence, the quantification of SA in AF or plasma of treated patients in the nmol/L range becomes very useful. The method utilizes 13C5-SA as an internal standard and a three-step sample treatment consisting of oximation, solvent extraction and TMCS derivatization. The assay was validated by recording the ion intensities of m/z 620 for SA and m/z 625 for ISTD in order to demonstrate the precision of measurements, the linearity of the method, limit of quantification and detection (LOQ and LOD), specificity, accuracy, as well as metabolite stability. Values for the intra-day assays ranged from 0.2 to 3.2% while values for the inter-day assays ranged from 1.9 to 5.6% confirming that the method has good precision. A calibration plot using SA detected by GC/MS gave excellent linearity with a correlation coefficient of 0.999 over the injected concentration range of 5-2000 nmol/L. LOQ and LOD were 3 and 1 nmol/L, respectively. The usefulness of this method was demonstrated by SA quantification in an AF sample of an affected fetus and in plasma of patients treated with NTBC. The results demonstrate that this novel GC/MS method may be a valuable tool for metabolic evaluation and clinical use.

A Metabolic Signature of Mitochondrial Dysfunction Revealed through a Monogenic Form of Leigh Syndrome

A decline in mitochondrial respiration represents the root cause of a large number of inborn errors of metabolism. It is also associated with common age-associated diseases and the aging process. To gain insight into the systemic, biochemical consequences of respiratory chain dysfunction, we performed a case-control, prospective metabolic profiling study in a genetically homogenous cohort of patients with Leigh syndrome French Canadian variant, a mitochondrial respiratory chain disease due to loss-of-function mutations in LRPPRC. We discovered 45 plasma and urinary analytes discriminating patients from controls, including classic markers of mitochondrial metabolic dysfunction (lactate and acylcarnitines), as well as unexpected markers of cardiometabolic risk (insulin and adiponectin), amino acid catabolism linked to NADH status (α-hydroxybutyrate), and NAD(+) biosynthesis (kynurenine and 3-hydroxyanthranilic acid). Our study identifies systemic, metabolic pathway derangements that can lie downstream of primary mitochondrial lesions, with implications for understanding how the organelle contributes to rare and common diseases.

Clinical heterogeneity in ethylmalonic encephalopathy.

Ethylmalonic encephalopathy is a recently described inborn error of metabolism characterized clinically by developmental delay and regression, recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhea. We describe monochorionic twins presenting with hypotonia in infancy and diagnosed with ethylmalonic encephalopathy on the basis of biochemical findings. They are compound heterozygote for missense mutations in ETHE1. Magnetic resonance imaging changes affecting the white matter, corpus callosum, and basal ganglia were seen in both patients. At 10 years of age, they have severe axial hypotonia but never displayed petechiae, orthostatic acrocyanosis, or chronic diarrhea. Their clinical courses differ markedly; one had an episode of coma when she was 3 years old and now has spastic quadraparesis and cannot speak. The other can freely use her upper extremities, her pyramidal syndrome being mostly limited to the lower extremities, and can speak 2 languages. These patients illustrate the clinical heterogeneity of ethylmalonic encephalopathy, even in monochorionic twins.

Mechanism of insulin-stimulated clearance of plasma nonesterified fatty acids in humans.

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<0.05). In contrast, plasma linoleate appearance, as expected, was increased by HI independently of insulin level (P<0.02). Plasma palmitate clearance during HINS/SAL was higher than LINS/SAL and LINS/HI (P<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.

On the suppression of plasma nonesterified fatty acids by insulin during enhanced intravascular lipolysis in humans.

During the fasting state, insulin reduces nonesterified fatty acid (NEFA) appearance in the systemic circulation mostly by suppressing intracellular lipolysis in the adipose tissue. In the postprandial state, insulin may also control NEFA appearance through enhanced trapping into the adipose tissue of NEFA derived from intravascular triglyceride lipolysis. To determine the contribution of suppression of intracellular lipolysis in the modulation of plasma NEFA metabolism by insulin during enhanced intravascular triglyceride lipolysis, 10 healthy nonobese subjects underwent pancreatic clamps at fasting vs. high physiological insulin level with intravenous infusion of heparin plus Intralipid. Nicotinic acid was administered orally during the last 2 h of each 4-h clamp to inhibit intracellular lipolysis and assess insulin's effect on plasma NEFA metabolism independently of its effect on intracellular lipolysis. Stable isotope tracers of palmitate, acetate, and glycerol were used to assess plasma NEFA metabolism and total triglyceride lipolysis in each participant. The glycerol appearance rate was similar during fasting vs. high insulin level, but plasma NEFA levels were significantly lowered by insulin. Nicotinic acid significantly blunted the insulin-mediated suppression of plasma palmitate appearance and oxidation rates by approximately 60 and approximately 70%, respectively. In contrast, nicotinic acid did not affect the marked stimulation of palmitate clearance by insulin. Thus most of the insulin-mediated reduction of plasma NEFA appearance and oxidation can be explained by suppression of intracellular lipolysis during enhanced intravascular triglyceride lipolysis in healthy humans. Our results also suggest that insulin may affect plasma NEFA clearance independently of the suppression of intracellular lipolysis.