Background
Lysosomal storage diseases (LSDs) are inborn errors of metabolism resulting from 50 different inherited disorders. The increasing availability of treatments and the importance of early ...intervention have stimulated newborn screening (NBS) to diagnose LSDs and permit early intervention to prevent irreversible impairment or severe disability. We present our experience screening newborns in North East Italy to identify neonates with Mucopolysaccharidosis type I (MPS I) and Pompe, Fabry, and Gaucher diseases.
Methods
Activities of acid β-glucocerebrosidase (ABG; Gaucher), acid α-glucosidase (GAA; Pompe), acid α-galactosidase (GLA; Fabry), and acid α-L-iduronidase (IDUA; MPS-I) in dried blood spots (DBS) from all newborns during a 17-month period were determined by multiplexed tandem mass spectrometry (MS/MS) using the NeoLSD
®
assay system. Enzymatic activity cutoff values were determined from 3500 anonymous newborn DBS. In the screening study, samples were retested if the value was below cutoff and a second spot was requested, with referral for confirmatory testing and medical evaluation if a low value was obtained.
Results
From September 2015 to January 2017, 44,411 newborns were screened for the four LSDs. We recalled 40 neonates (0.09%) for collection of a second DBS. Low activity was confirmed in 20, who had confirmatory testing. Ten of 20 had pathogenic mutations: two Pompe, two Gaucher, five Fabry, and one MPS-I. The incidences of Pompe and Gaucher diseases were similar (1/22,205), with Fabry disease the most frequent (1/8882) and MPS-I the rarest (1/44411). The combined incidence of the four disorders was 1/4411 births.
Conclusions
Simultaneously determining multiple enzyme activities by MS/MS, with a focus on specific biochemical markers, successfully detected newborns with LSDs. The high incidence of these disorders supports this screening program.
Lysosphingolipids (LysoSLs) are derivatives of sphingolipids which have lost the amide-linked acyl chain. More recently, LysoSLs have been identified as storage compounds in several sphingolipidoses, ...including Gaucher, Fabry and Niemann-Pick diseases. To date, different methods have been developed to measure each individual lysosphingolipid in plasma. This report describes a rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of several LysoSLs in plasma.
We analyzed the following compounds: hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM) and lysosphingomyelin-509 (LysoSM-509). The sample preparation requires only 100 μL of plasma and consists of an extraction with a mixture of MeOH/acetone/H2O (45:45:10, v/v).
The method validation showed high sensitivity, an excellent accuracy and precision. Reference ranges were determined in healthy adult and pediatric population. The results demonstrate that the LC-MS/MS method can quantify different LysoSLs and can be used to identify patients with Fabry (LysoGb3), Gaucher and Krabbe (HexSph) diseases, prosaposine deficiency (LysoGb3 and HexSph), and Niemann-Pick disease types A/B and C (LysoSM and LysoSM-509).
This LC-MS/MS method allows a rapid and simultaneous quantification of LysoSLs and is useful as a biochemical diagnostic tool for sphingolipidoses.
Dysfunction of mitochondrial respiration is an increasingly recognized cause of isolated hypertrophic cardiomyopathy. To gain insight into the genetic origin of this condition, we used ...next-generation exome sequencing to identify mutations in MTO1, which encodes mitochondrial translation optimization 1. Two affected siblings carried a maternal c.1858dup (p.Arg620Lysfs∗8) frameshift and a paternal c.1282G>A (p.Ala428Thr) missense mutation. A third unrelated individual was homozygous for the latter change. In both humans and yeast, MTO1 increases the accuracy and efficiency of mtDNA translation by catalyzing the 5-carboxymethylaminomethylation of the wobble uridine base in three mitochondrial tRNAs (mt-tRNAs). Accordingly, mutant muscle and fibroblasts showed variably combined reduction in mtDNA-dependent respiratory chain activities. Reduced respiration in mutant cells was corrected by expressing a wild-type MTO1 cDNA. Conversely, defective respiration of a yeast mto1Δ strain failed to be corrected by an Mto1Pro622∗ variant, equivalent to human MTO1Arg620Lysfs∗8, whereas incomplete correction was achieved by an Mto1Ala431Thr variant, corresponding to human MTO1Ala428Thr. The respiratory yeast phenotype was dramatically worsened in stress conditions and in the presence of a paromomycin-resistant (PR) mitochondrial rRNA mutation. Lastly, in vivo mtDNA translation was impaired in the mutant yeast strains.
Background Lysosphingolipids, the N-deacylated forms of sphingolipids, have been identified as potential biomarkers of several sphingolipidoses, such as Gaucher, Fabry, Krabbe and Niemann-Pick ...diseases and in GM1 and GM2 gangliosidoses. To date, different methods have been developed to measure various lysosphingolipids (LysoSLs) in plasma. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a simultaneous quantification of LysoSLs (HexSph, LysoGb3, LysoGM1, LysoGM2, LysoSM and LysoSM509) in dried blood spot (DBS). This LC-MS/MS method was used to compare the levels of LysoSLs in DBS and plasma in both affected patients and healthy controls. Methods Lysosphingolipids were extracted from a 3.2 mm diameter DBS with a mixture of methanol:acetonitrile:water (80:15:5, v/v) containing internal stable isotope standards. Chromatographic separation was performed using a C18 column with a gradient of water and acetonitrile both with 0.1% formic acid in a total run time of 4 min. The compounds were detected in the positive ion mode electrospray ionization (ESI)-MS/MS by multiple reaction monitoring (MRM). Results The method was validated on DBS to demonstrate specificity, linearity, lowest limit of quantification, accuracy and precision. The reference ranges were determined in pediatric and adult populations. The elevated levels of LysoSLs were identified in Gaucher disease (HexSph), Fabry disease (LysoGb3), prosaposin deficiency (HexSph and LysoGb3) and Niemann-Pick disease types A/B and C (LysoSM and LysoSM509). The correlation in the levels between DBS and plasma was excellent for LysoGb3 and HexSph but poor for LysoSM and LysoSM509. Conclusions Despite the fact that plasma LysoSLs determination remains the gold standard, our LC-MS/MS method allows a rapid and reliable quantification of lysosphingolipids in DBS. The method is a useful tool for the diagnosis of different sphingolipidoses except for Niemann-Pick type C.
Glutaric aciduria type I (synonym, glutaric acidemia type I) is a rare organic aciduria. Untreated patients characteristically develop dystonia during infancy resulting in a high morbidity and ...mortality. The neuropathological correlate is striatal injury which results from encephalopathic crises precipitated by infectious diseases, immunizations and surgery during a finite period of brain development, or develops insidiously without clinically apparent crises. Glutaric aciduria type I is caused by inherited deficiency of glutaryl-CoA dehydrogenase which is involved in the catabolic pathways of L-lysine, L-hydroxylysine and L-tryptophan. This defect gives rise to elevated glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutarylcarnitine which can be detected by gas chromatography/mass spectrometry (organic acids) or tandem mass spectrometry (acylcarnitines). Glutaric aciduria type I is included in the panel of diseases that are identified by expanded newborn screening in some countries. It has been shown that in the majority of neonatally diagnosed patients striatal injury can be prevented by combined metabolic treatment. Metabolic treatment that includes a low lysine diet, carnitine supplementation and intensified emergency treatment during acute episodes of intercurrent illness should be introduced and monitored by an experienced interdisciplinary team. However, initiation of treatment after the onset of symptoms is generally not effective in preventing permanent damage. Secondary dystonia is often difficult to treat, and the efficacy of available drugs cannot be predicted precisely in individual patients. The major aim of this revision is to re-evaluate the previous diagnostic and therapeutic recommendations for patients with this disease and incorporate new research findings into the guideline.
The gold standard treatment for phenylketonuria (PKU) is a lifelong low-phenylalanine (Phe) diet supplemented with Phe-free protein substitutes. Adherence to therapy becomes difficult after ...childhood. Supplementing with large neutral amino acids (LNAAs) has been proposed as an alternative medication to Phe-free protein substitutes (i.e., amino acid mixtures). The aim of this study was to evaluate adherence to therapy and quality of life (QoL) in a cohort of sub-optimally controlled adult PKU patients treated with a new LNAA formulation. Twelve patients were enrolled in a 12-month-trial of slow-release LNAAs (1g/kg/day) plus a Phe-restricted diet. Medication adherence was measured with the Morisky Green Levine Medication Adherence Scale; the QoL was measured using the phenylketonuria-quality of life (PKU-QoL) questionnaire. Phe, tyrosine (Tyr) levels, and Phe/Tyr ratios were measured fortnightly. Before treatment, 3/12 patients self-reported a 'medium' adherence to medication and 9/12 reported a low adherence; 60% of patients reported a full adherence over the past four weeks. After 12 months of LNAA treatment, all patients self-reported a high adherence to medication, with 96% reporting a full adherence. Phe levels remained unchanged, while Tyr levels increased in most patients. The Phy/Tyr ratio decreased. All patients had a significant improvement in the QoL. LNAAs may give patients a further opportunity to improve medication adherence and, consequently, their QoL.
Phenylketonuria (PKU) is a rare inborn error of metabolism affecting the catabolism of phenylalanine (Phe). To date, findings regarding health-related quality of life (HRQoL) in adults with ...early-treated classical PKU are discrepant. Moreover, little is known about metabolic, demographic, and cognitive factors associated with HRQoL. Hence, we aimed to investigate HRQoL and its association with demographic, metabolic, and cognitive characteristics in a large European sample of adults with early-treated classical PKU. This cross-sectional study included 124 adults with early-treated classical PKU from Hungary, Italy, Spain, Switzerland, and Turkey. All participants prospectively completed the PKU quality of life questionnaire (PKU-QoL), a questionnaire specifically designed to evaluate the impact of PKU and its treatment on HRQoL in individuals with PKU. In addition, information about Phe levels (concurrent and past year), demographic (age and sex), and cognitive variables (intelligence quotient, IQ) were collected. Most domains revealed little or no impact of PKU on HRQoL and more than three-quarters of the patients rated their health status as good, very good, or excellent. Nevertheless, some areas of concern for patients were identified. Patients were worried about the guilt that they experience if they do not adhere to the dietary protein restriction and they were most concerned about high Phe levels during pregnancy. Further, tiredness was the most affected symptom, and the supplements' taste was considered a main issue for individuals with PKU. The overall impact of PKU on HRQoL was higher in women (U = 1315.5, p = .012) and in adults with a lower IQ (r.sub.s = - 0.448, p = .005). The overall impact of dietary protein restriction was higher in adults with higher concurrent Phe levels (r.sub.s = 0.272, p = .007) and higher Phe levels during the past year (r.sub.s = 0.280, p = .009). The impact of PKU on most domains assessed in the PKU-QoL was considered to be low. These results likely reflect the successful implementation of the newborn screening resulting in the prevention of severe adverse long-term outcomes. However, a particular clinical focus should be given to patients with lower IQ, higher Phe levels, and women, as these variables were associated with a lower HRQoL.
The standard treatment for phenylketonuria (PKU) is a lifelong low-phenylalanine (Phe) diet, supplemented with Phe-free protein substitutes; however, adult patients often show poor adherence to ...therapy. Alternative treatment options include the use of large neutral amino acids (LNAA). The aim of this study was to determine the Phe, tyrosine (Tyr), and Phe/Tyr ratio in a cohort of sub-optimally controlled adult patients with classical PKU treated with a new LNAA formulation. Twelve patients received a Phe-restricted diet plus a slow-release LNAA product taken three times per day, at a dose of 1 g/kg body weight (mean 0.8 ± 0.24 g/kg/day), over a 12-month period. The product is in a microgranulated formulation, which incorporates all amino acids and uses sodium alginate as a hydrophilic carrier to prolong its release. This LNAA formulation provides up to 80% of the total protein requirement, with the rest of the protein supplied by natural food. Patients had fortnightly measurements of Phe and Tyr levels over a 12-month period after the introduction of LNAA. All patients completed the 12-month treatment period. Overall, adherence to the new LNAA tablets was very good compared with a previous amino acid mixture, for which taste was a major complaint by patients. Phe levels remained unchanged (
= 0.0522), and Tyr levels increased (
= 0.0195). Consequently, the Phe/Tyr ratio decreased significantly (
< 0.05) in the majority of patients treated. In conclusion, LNAA treatment increases Tyr levels in sub-optimally controlled adult PKU patients, while offering the potential to improve their adherence to treatment.
Objectives Mucopolysaccharidosis type I (MPS I) was added to our expanded screening panel in 2015. Since then, 127,869 newborns were screened by measuring α-L-iduronidase (IDUA) enzyme activity with ...liquid chromatography tandem mass spectrometry (LC-MS/MS). High false positives due to frequent pseudodeficiency alleles prompted us to develop a second-tier test to quantify glycosaminoglycan (GAG) levels in dried blood spot (DBS). Methods Heparan-sulfate (HS) and dermatan-sulfate (DS) were measured with LC-MS/MS after methanolysis. DBSs were incubated with methanolic-HCl 3 N at 65 °C for 45 min. Chromatographic separation used an amide column with a gradient of acetonitrile and water with 10 mM ammonium acetate in a 9-min run. The method was validated for specificity, linearity, lower limit of quantification (LOQ), accuracy and precision. Results Intra- and inter-day coefficients of variation were <15% for both metabolites. Reference values in 40 healthy newborns were: HS mean 1.0 mg/L, 0-3.2; DS mean 1.5 mg/L, 0.5-2.7). The two confirmed newborn MPS I patients had elevated HS (4.9-10.4 mg/L, n.v. <3.2) and DS (7.4-8.8 mg/L, n.v. <2.7). Since its introduction in February 2019, the second-tier test reduced the recall rate from 0.046% to 0.006%. Among 127,869 specimens screened, the incidence was 1:63,935 live births. Both patients started enzyme replacement therapy (ERT) within 15 days of birth and one of them received allogenic hematopoietic stem cell transplantation (HSCT) at ht age of 6 months. Conclusions GAGs in DBS increased the specificity of newborn screening for MPS I by reducing false-positives due to heterozygosity or pseudodeficiency. Early diagnosis and therapeutical approach has improved the outcome of our patients with MPS I.
Gaucher disease is the most common of the lysosomal storage diseases. It presents a wide phenotypic continuum, in which one may identify the classically described phenotypes, including type 1 form ...with visceral involvement, type 2 acute neuropathic early‐infantile form, and type 3 subacute neuronopathic form. At the most severe end there is the perinatal form with onset in utero or during the neonatal period. The very few reported cases of neonatal onset Gaucher disease presented high and early mortality, due to neurological or visceral involvement, including liver failure. We report our experience treating a patient with the neonatal form of Gaucher disease who presented at birth with thrombocytopenia, hepatosplenomegaly and cholestasis. Despite early enzyme replacement therapy, liver disease was progressive. Liver biopsy showed hepatocellular giant‐cell transformation, a nonspecific finding consistent with inflammation. The lack of response to enzyme replacement therapy and the microscopic findings suggested that mechanisms apart from substrate accumulation and Gaucher cells may play a role in the hepatic pathogenesis in Gaucher disease. An attempt to use corticosteroids at the age of 3 months resulted in a dramatic improvement in liver function and resulted in long‐term survival. The patient is alive and 2 years old at this writing. Our case suggests that inflammatory processes may be important in the early pathogenesis of Gaucher disease and that early use of corticosteroids may open the way to a new therapeutic approach.
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