Non-Hodgkin lymphoma in a kidney transplanted patient with methylmalonic acidemia: Metabolic susceptibility and the role of immunosuppression.

Methylmalonic acidemia cblB type (MMA cblB) is an autosomal recessive inborn error of amino acid metabolism that results in impaired synthesis of adenosylcobalamin, a cofactor of methylmalonyl-CoA mutase. It presents with episodes of coma, vomiting, hypotonia, metabolic acidosis, and hyperammonemia. End-stage kidney disease is a long-term complication. Treatments include vitamin B12 supplementation, L-carnitine, and a low-protein diet. Liver, kidney, or combined liver-kidney transplantations are promising options, but they are not without complications. We report a patient suffering from MMA cblB who developed end-stage kidney disease at 18 years of age. Kidney transplantation allowed him to recover normal kidney function and good metabolic control. Unfortunately, after two decades, he developed non-Hodgkin lymphoma and severe chemotherapy toxicity which led to his death. The risk of lymphoproliferative diseases is known to increase after solid organ transplantation. However, in MMA, factors including mitochondrial dysfunction and oncometabolites, may further increase the risk of malignancy and drug toxicity. Our report highlights the importance of considering the increased risk of cancer in long-term follow-up of MMA cblB patients, especially after solid organ transplantation. Moreover, when chemotherapy is needed, the increased risk of toxicity and metabolic decompensation should be considered and monitored.

Health-related quality of life in a european sample of adults with early-treated classical PKU.

BACKGROUND: 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. RESULTS: 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 (rs = - 0.448, p = .005). The overall impact of dietary protein restriction was higher in adults with higher concurrent Phe levels (rs = 0.272, p = .007) and higher Phe levels during the past year (rs = 0.280, p = .009). CONCLUSION: 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.

Variant in the allosteric domain of CPS1 protein associated with effectiveness of N-carbamoyl glutamate therapy in neonatal onset CPS1 deficiency.

OBJECTIVES: Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a severe urea cycle disorder. Patients can present with hyperammonemic coma in the first days of life. Treatment includes nitrogen scavengers, reduced protein intake and supplementation with L-arginine and/or L-citrulline. N-carbamoyl glutamate (NCG) has been hypothesized to stimulate the residual CPS1 function, although only few patients are reported. CASE PRESENTATION: We report a patient with neonatal-onset CPS1 deficiency who received NCG in association with nitrogen scavenger and L-citrulline. The patient carried the novel variants CPS1-c.2447A>G p.(Gln816Arg) and CPS1-c.4489T>C p.(Tyr1497His). The latter is localized in the C-terminal allosteric domain of the protein, and is implicated in the binding of the natural activator N-acetyl-L-glutamate. NCG therapy was effective in controlling ammonia levels, allowing to increase the protein intake. CONCLUSIONS: Our data show that the response to NCG can be indicated based on the protein structure. We hypothesize that variants in the C-terminal domain may be responsive to NCG therapy.

Newborn Screening for Fabry Disease: Current Status of Knowledge.

Fabry disease is an X-linked progressive lysosomal disorder, due to α-galactosidase A deficiency. Patients with a classic phenotype usually present in childhood as a multisystemic disease. Patients presenting with the later onset subtypes have cardiac, renal and neurological involvements in adulthood. Unfortunately, the diagnosis is often delayed until the organ damage is already irreversibly severe, making specific treatments less efficacious. For this reason, in the last two decades, newborn screening has been implemented to allow early diagnosis and treatment. This became possible with the application of the standard enzymology fluorometric method to dried blood spots. Then, high-throughput multiplexable assays, such as digital microfluidics and tandem mass spectrometry, were developed. Recently DNA-based methods have been applied to newborn screening in some countries. Using these methods, several newborn screening pilot studies and programs have been implemented worldwide. However, several concerns persist, and newborn screening for Fabry disease is still not universally accepted. In particular, enzyme-based methods miss a relevant number of affected females. Moreover, ethical issues are due to the large number of infants with later onset forms or variants of uncertain significance. Long term follow-up of individuals detected by newborn screening will improve our knowledge about the natural history of the disease, the phenotype prediction and the patients' management, allowing a better evaluation of risks and benefits of the newborn screening for Fabry disease.

Long-term follow-up of a patient with neonatal form of Gaucher disease.

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.

Unusual Evolution of Hypertrophic Cardiomyopathy in Non-Compaction Myocardium in a Pompe Disease Patient.

Classic infantile Pompe disease is characterized by a severe phenotype with cardiomyopathy and hypotonia. Cardiomyopathy is generally hypertrophic and rapidly regresses after enzyme replacement therapy. In this report, for the first time, we describe a patient with infantile Pompe disease and hypertrophic cardiomyopathy that evolved into non-compaction myocardium after treatment. The male newborn had suffered since birth with hypertrophic cardiomyopathy and heart failure. He was treated with standard enzyme replacement therapy (ERT) (alglucosidase alfa) and several immunomodulation cycles due to the development of anti-ERT antibodies, without resolution of the hypertrophic cardiomyopathy. At the age of 2.5 years, he was treated with a new combination of ERT therapy (cipaglucosidase alfa) and a chaperone (miglustat) for compassionate use. After 1 year, the cardiac hypertrophy was resolved, but it evolved into non-compaction myocardium. Non-compaction cardiomyopathy is often considered to be a congenital, primitive cardiomyopathy, due to an arrest of compaction of the myocardium wall during the embryonal development. Several genetic causes have been identified. We first describe cardiac remodeling from hypertrophic cardiomyopathy to a non-compaction form in a patient with infantile Pompe disease treated with a new ERT. This has important implications both for the monitoring of Pompe disease patients and for the understanding of the pathophysiological basis of non-compaction myocardium.

Light and Shadows in Newborn Screening for Lysosomal Storage Disorders: Eight Years of Experience in Northeast Italy.

In the last two decades, the development of high-throughput diagnostic methods and the availability of effective treatments have increased the interest in newborn screening for lysosomal storage disorders. However, long-term follow-up experience is needed to clearly identify risks, benefits and challenges. We report our 8-year experience of screening and follow-up on about 250,000 neonates screened for four lysosomal storage diseases (Pompe disease, mucopolysaccharidosis type I, Fabry disease, Gaucher disease), using the enzyme activity assay by tandem mass spectrometry, and biomarker quantification as a second-tier test. Among the 126 positive newborns (0.051%), 51 infants were confirmed as affected (positive predictive value 40%), with an overall incidence of 1:4874. Of these, three patients with infantile-onset Pompe disease, two with neonatal-onset Gaucher disease and four with mucopolysaccharidosis type I were immediately treated. Furthermore, another four Gaucher disease patients needed treatment in the first years of life. Our study demonstrates the feasibility and effectiveness of newborn screening for lysosomal storage diseases. Early diagnosis and treatment allow the achievement of better patient outcomes. Challenges such as false-positive rates, the diagnosis of variants of uncertain significance or late-onset forms and the lack of treatment for neuronopathic forms, should be addressed.

Newborn screening for Pompe disease in Italy: Long-term results and future challenges.

Pompe disease (PD) is a progressive neuromuscular disorder caused by a lysosomal acid α-glucosidase (GAA) deficiency. Enzymatic replacement therapy is available, but early diagnosis by newborn screening (NBS) is essential for early treatment and better outcomes, especially with more severe forms. We present results from 7 years of NBS for PD and the management of infantile-onset (IOPD) and late-onset (LOPD) patients, during which we sought candidate predictive parameters of phenotype severity at baseline and during follow-up. We used a tandem mass spectrometry assay for α-glucosidase activity to screen 206,741 newborns and identified 39 positive neonates (0.019%). Eleven had two pathogenic variants of the GAA gene (3 IOPD, 8 LOPD); six carried variants of uncertain significance (VUS). IOPD patients were treated promptly and had good outcomes. LOPD and infants with VUS were followed; all were asymptomatic at the last visit (mean age 3.4 years, range 0.5-5.5). Urinary glucose tetrasaccharide was a useful and biomarker for rapidly differentiating IOPD from LOPD and monitoring response to therapy during follow-up. Our study, the largest reported to date in Europe, presents data from longstanding NBS for PD, revealing an incidence in North East Italy of 1/18,795 (IOPD 1/68,914; LOPD 1/25,843), and the absence of mortality in IOPD treated from birth. In LOPD, rigorous long-term follow-up is needed to evaluate the best time to start therapy. The high pseudodeficiency frequency, ethical issues with early LOPD diagnosis, and difficulty predicting phenotypes based on biochemical parameters and genotypes, especially in LOPD, need further study.

Galactose epimerase deficiency: lessons from the GalNet registry.

BACKGROUND: Galactose epimerase (GALE) deficiency is a rare hereditary disorder of galactose metabolism with only a few cases described in the literature. This study aims to present the data of patients with GALE deficiency from different countries included through the Galactosemia Network to further expand the existing knowledge and review the current diagnostic strategy, treatment and follow-up of this not well characterized entity. METHODS: Observational study collecting medical data from December 2014 to April 2022 of 22 not previously reported patients from 14 centers in 9 countries. Patients were classified as generalized or non-generalized based on their genotype, enzyme activities in different tissues and/or clinical picture and professional judgment of the treating physician. RESULTS: In total 6 patients were classified as generalized and 16 as non-generalized. In the generalized group, acute neonatal illness was reported in 3, cognitive and developmental delays were present in 5 and hearing problems were reported in 3. Four generalized patients were homozygous for the genetic variant NM_001008216.2:c.280G > A (p.Val94Met). In the non-generalized group, no clearly related symptoms were found. Ten novel genetic variants were reported in this study population. CONCLUSION: The phenotypic spectrum of GALE deficiency ranges from asymptomatic to severe. The generalized patients have a phenotype that is in line with the 9 described cases in the literature and prescribing dietary interventions is the cornerstone for treatment. In the non-generalized group, treatment advice is more difficult. To be able to offer proper counseling, in addition to red blood cell enzyme activity, genetic studies, transferrin glycoform analysis and enzymatic measurements in fibroblasts are recommended. Due to lack of facilities, additional enzymatic testing is not common practice in many centers nor a tailored long-term follow-up is performed.

Bone disease in early detected Gaucher Type I disease: A case report.

Gaucher disease (GD) is a lysosomal disorder characterized by the storage of glucosylceramide in macrophages ("Gaucher cells"), particularly in the spleen, liver, and bone marrow. The most common phenotype, GD type 1, usually presents with hepatosplenomegaly, cytopenias, and sometimes bone involvement at variable age. Enzyme replacement therapy (ERT) is available and effective, but some severe manifestations are irreversible (e.g., osteonecrosis), so that early treatment is crucial. We describe a 4-year-old Albanian male with GD type 1, diagnosed through newborn screening (NBS), presented during follow up with multiple osteonecrotic areas in both femurs. He had no other symptoms or signs of disease, except for increasing of lyso-Gb1 biomarker. Early initiation of ERT allowed a partial improvement of bone lesions. Our case highlights the importance of NBS for GD and of close follow-up of presymptomatic patients, especially if biomarker levels are increasing. In the absence of NBS, GD should be considered in patients who present with bone lesions, also isolated. Early diagnosis and treatment improve the course of disease and avoid irreversible sequelae.

A new strategy of desensitization in mucopolysaccharidosis type II disease treated with idursulfase therapy: A case report and review of the literature.

Mucopolysaccharidosis type II (MPS II) is a multisystemic lysosomal storage disorder caused by deficiency of the iduronate 2-sulfatase enzyme. Currently, enzyme replacement therapy (ERT) with recombinant idursulfase is the main treatment available to decrease morbidity and improve quality of life. However, infusion-associated reactions (IARs) are reported and may limit access to treatment. When premedication or infusion rate reductions are ineffective for preventing IARs, desensitization can be applied. To date, only two MPS II patients are reported to have undergone desensitization. We report a pediatric patient with recurrent IARs during infusion successfully managed with gradual desensitization. Our protocol started at 50% of the standard dosage infused at concentrations from 0.0006 to 0.06 mg/ml on weeks 1 and 2, followed by 75% of the standard dosage infused at concentrations from 0.0009 to 0.09 mg/ml on weeks 3 and 4, and full standard dosage thereafter, infused at progressively increasing concentrations until the standard infusion conditions were reached at 3 months. Our experience can be used in the management of MPS II patients presenting IARs to idursulfase infusion, even when general preventive measures are already administered.

Newborn screening of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I), a lysosomal storage disease caused by a deficiency of α-L-iduronidase, leads to storage of the glycosaminoglycans, dermatan sulfate and heparan sulfate. Available therapies include enzyme replacement and hematopoietic stem cell transplantation. In the last two decades, newborn screening (NBS) has focused on early identification of the disorder, allowing early intervention and avoiding irreversible manifestations. Techniques developed and optimized for MPS I NBS include tandem mass-spectrometry, digital microfluidics, and glycosaminoglycan quantification. Several pilot studies have been conducted and screening programs have been implemented worldwide. NBS for MPS I has been established in Taiwan, the United States, Brazil, Mexico, and several European countries. All these programs measure α-L-iduronidase enzyme activity in dried blood spots, although there are differences in the analytical strategies employed. Screening algorithms based on published studies are discussed. However, some limitations remain: one is the high rate of false-positive results due to frequent pseudodeficiency alleles, which has been partially solved using post-analytical tools and second-tier tests; another involves the management of infants with late-onset forms or variants of uncertain significance. Nonetheless, the risk-benefit ratio is favorable. Furthermore, long-term follow-up of patients detected by neonatal screening will improve our knowledge of the natural history of the disease and inform better management.

Newborn Screening for Fabry Disease in Northeastern Italy: Results of Five Years of Experience.

Fabry disease (FD) is a progressive multisystemic lysosomal storage disease. Early diagnosis by newborn screening (NBS) may allow for timely treatment, thus preventing future irreversible organ damage. We present the results of 5.5 years of NBS for FD by α-galactosidase A activity and globotriaosylsphingosine (lyso-Gb3) assays in dried blood spot through a multiplexed MS/MS assay. Furthermore, we report our experience with long-term follow-up of positive subjects. We screened more than 170,000 newborns and 22 males were confirmed to have a GLA gene variant, with an incidence of 1:7879 newborns. All patients were diagnosed with a variant previously associated with the later-onset phenotype of FD or carried an unclassified variant (four patients) or the likely benign p.Ala143Thr variant. All were asymptomatic at the last visit. Although lyso-Gb3 is not considered a reliable second tier test for newborn screening, it can simplify the screening algorithm when its levels are elevated at birth. After birth, plasma lyso-Gb3 is a useful marker for non-invasive monitoring of all positive patients. Our study is the largest reported to date in Europe, and presents data from long-term NBS for FD that reveals the current incidence of FD in northeastern Italy. Our follow-up data describe the early disease course and the trend of plasma lyso-Gb3 during early childhood.

Nutrition in adult patients with selected lysosomal storage diseases.

Lysosomal storage disorders (LSDs) are a group of clinically heterogeneous disorders affecting the function of lysosomes and are characterized by an accumulation of undigested substrates within several cell types. In recent years there have been substantial advances in supportive care and drug treatment for some LSDs, leading to improved patient survival, as seen in Gaucher, Pompe and Fabry disease and some Mucopolysaccharidoses; however, many symptoms still persist. Thus it is now even more important to improve patients' quality of life and reduce symptoms and comorbidities. One potential way of achieving this goal is through adjunct nutritional therapy, which is challenging as patients may be overweight with associated consequences, or malnourished, or underweight. Furthermore, drugs used to treat LSDs can modify the metabolic status and needs of patients. There are currently not enough data to make specific dietary recommendations for individual LSDs; however, suggestions can be made for managing clinical manifestations of the diseases, as well as treatment-associated adverse events. The metabolic and nutritional status of adult patients must be regularly assessed and individualized dietary plans may be created to cater to a patient's specific needs. Damage to the autophagic process is a common feature in LSDs that is potentially sensitive to dietary manipulation and needs to be assessed in clinical studies.

The Impact of a Slow-Release Large Neutral Amino Acids Supplement on Treatment Adherence in Adult Patients with Phenylketonuria.

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.

The combined use of enzyme activity and metabolite assays as a strategy for newborn screening of mucopolysaccharidosis type I.

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.

Oxygen and nitrite reduction by heme-deficient sulphite oxidase in a patient with mild sulphite oxidase deficiency.

Isolated sulphite oxidase deficiency (iSOD) is an autosomal recessive inborn error in metabolism characterised by accumulation of sulphite, which leads to death in early infancy. Sulphite oxidase (SO) is encoded by the SUOX gene and forms a heme- and molybdenum-cofactor-dependent enzyme localised in the intermembrane space of mitochondria. Within SO, both cofactors are embedded in two separated domains, which are linked via a flexible 11 residue tether. The two-electron oxidation of sulphite to sulphate occurs at the molybdenum active site. From there, electrons are transferred via two intramolecular electron transfer steps (IETs) via the heme cofactor and to the physiologic electron acceptor cytochrome c. Previously, we reported nitrite and oxygen to serve as alternative electron acceptors at the Moco active site, thereby overcoming IET within SO. Here, we present evidence for these reactions to occur in an iSOD patient with an unusual mild disease representation. In the patient, a homozygous c.427C>A mutation within the SUOX gene leads to replacement of the highly conserved His143 to Asn. The affected His143 is one of two heme-iron-coordinating residues within SO. We demonstrate, that the H143N SO variant fails to bind heme in vivo leading to the elimination of SO-dependent cytochrome c reduction in mitochondria. We show, that sulphite oxidation at the Moco domain is unaffected in His143Asn SO variant and demonstrate that nitrite and oxygen are able to serve as electron acceptors for sulphite-derived electrons in cellulo. As result, the patient H143N SO variant retains residual sulphite oxidising activity thus ameliorating iSOD progression.

Large Neutral Amino Acid Therapy Increases Tyrosine Levels in Adult Patients with Phenylketonuria: A Long-Term Study.

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 (p = 0.0522), and Tyr levels increased (p = 0.0195). Consequently, the Phe/Tyr ratio decreased significantly (p < 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.

Plasma and dried blood spot lysosphingolipids for the diagnosis of different sphingolipidoses: a comparative study.

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.