Living with phenylketonuria in adulthood: the PKU ATTITUDE study

Dietary treatment is the cornerstone of therapy for phenylketonuria (PKU), but adherence to low- phenylalanine diet progressively decreases after adolescence. We designed a survey to characterize the dietary habits of Italian adult PKU patients and to identify psychological factors influencing disease perception and adherence to diet. Participants to the survey (n = 111; response rate 94%) were asked to complete a structured questionnaire. Patients appeared to have an altered perception and awareness of the disease. About 40% of them did not consider PKU a disease and, despite declaring regular monitoring of phenylalanine levels (85%), nearly half of them reported a high plasma value over the last 6 months (>600 μmol/L, 48%) or were unable to specify it (31%). Adherence to PKU diet was unsatisfactory, with increased consumption of natural protein sources and reduced daily use of amino-acid supplements (<4–5 times/day in 82% patients). In addition to the intrinsic characteristics of AA formula (palatability, ease of use), the most important factor influencing their consumption was the increased social pressure associated with their use (55%). Plasma phenylalanine periodical measurements (61%) and examinations at metabolic centers (49%) were considered relevant for compliance to diet. In Italian adult PKU patients dietary management was found to be inadequate, likely due to inappropriate perception and knowledge of the disease, and lack of awareness of the negative impact of poor metabolic control in adult life. Clinicians should consider implementing more intense and tailored educational measures, as well as structured transitional care processes

Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions.

TIMM50 is an essential component of the TIM23 complex, the mitochondrial inner membrane machinery that imports cytosolic proteins containing a mitochondrial targeting presequence into the mitochondrial inner compartment. Whole exome sequencing (WES) identified compound heterozygous pathogenic mutations in TIMM50 in an infant patient with rapidly progressive, severe encephalopathy. Patient fibroblasts presented low levels of TIMM50 and other components of the TIM23 complex, lower mitochondrial membrane potential, and impaired TIM23-dependent protein import. As a consequence, steady-state levels of several components of mitochondrial respiratory chain were decreased, resulting in decreased respiration and increased ROS production. Growth of patient fibroblasts in galactose shifted energy production metabolism toward oxidative phosphorylation (OxPhos), producing an apparent improvement in most of the above features but also increased apoptosis. Complementation of patient fibroblasts with TIMM50 improved or restored these features to control levels. Moreover, RNASEH1 and ISCU mutant fibroblasts only shared a few of these features with TIMM50 mutant fibroblasts. Our results indicate that mutations in TIMM50 cause multiple mitochondrial bioenergetic dysfunction and that functional TIMM50 is essential for cell survival in OxPhos-dependent conditions

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

Abstract 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

The management of transitional care of patients affected by phenylketonuria in Italy: Review and expert opinion

Phenylketonuria (PKU) is a metabolic inherited disorder in which transition from infancy to adult care is particularly difficult and not sufficiently regulated. According to the scientific literature, only few medical centers offer healthcare assistance for adult patients with PKU that are therefore still treated in pediatric settings. This generates psychological, emotional, and organizational discomfort among patients, leading them to discontinue the follow-up. European guidelines and national consensus documents underline this unmet need and the lack of practical recommendations for a structured transitional pathway in PKU. The aim of this review and expert opinion is to propose good practices for managing the transition period of PKU patients, based on the literature and the experience of a panel of Italian experts in PKU. The consensus of the experts was obtained through the administration of three rounds of surveys and one structured interview. The result is the first proposal of a pathway for an efficient transition of PKU patients. Key steps of the proposed pathway are the "a priori" planning involving the pediatric and adult teams, the acceptance of the patient and his/her family to the process, the preliminary definition of appropriate spaces in the structure, the organization of meetings with the joint team, and the appointment of a transition coordinator. For the first time, the involvement of decision makers and patient associations is proposed

New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease.

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease(WD) or the more benign cholesteryl ester storage disease (CESD). To identifythe molecular basis of the different phenotypes we have characterised the LALgene mutations in three new patients with LAL deficiency. A patient with WD washomozygote for a null allele Y303X. The other two patients, with CESD, presentedeither homozygosity for T267I or compound heterozygosity consisting of Q64R andan exon 8 donor splice site substitution (G→A in position–1). The mutants T267I and Q64R and the previously reported L273S, G66V,and H274Y CESD substitutions, overexpressed in stable clones, were found to befully glycosylated and show an enzymatic activity of 3–8% of that ofnormal LAL. On the other hand, the Δ254–277 mutant proteinderived from exon 8 skipping and the Y303X protein were totally inactive. Bytransient transfection of hybrid minigene constructs, the CESD G→A(–1) substitution resulted in partial exon inclusion, thus allowing theproduction of a small amount of normal LAL mRNA and hence of a functionalenzyme. In contrast, a G→Asubstitution observed in WD at position +1 of the same exon 8 donor siteresulted in complete exon skipping and the sole production of an inactiveΔ254–277 protein.In conclusion,LAL genotypes determine the level of residual enzymatic activity, thusexplaining the severity of the phenotype.—Pagani, F., R. Pariyarath, R.Garcia, C. Stuani, A. B. Burlina, G. Ruotolo, M. Rabusin, and F. E. Baralle. Newlysosomal acid lipase gene mutants explain the phenotype of Wolman disease andcholesteryl ester storage disease. J. Lipid Res. 1998. 39:1382–1388

Phenotypic characteristics of the p.Asn215Ser (p.N215S) GLA mutation in male and female patients with Fabry disease: A multicenter Fabry Registry study.

BackgroundThe p.Asn215Ser or p.N215S GLA variant has been associated with late-onset cardiac variant of Fabry disease.MethodsTo expand on the scarce phenotype data, we analyzed natural history data from 125 p.N215S patients (66 females, 59 males) enrolled in the Fabry Registry (NCT00196742) and compared it with data from 401 patients (237 females, 164 males) harboring mutations associated with classic Fabry disease. We evaluated interventricular septum thickness (IVST), left ventricular posterior wall thickness (LVPWT), estimated glomerular filtration rate and severe clinical events.ResultsIn p.N215S males, mildly abnormal mean IVST and LVPWT values were observed in patients aged 25-34 years, and values gradually increased with advancing age. Mean values were similar to those of classic males. In p.N215S females, these abnormalities occurred primarily in patients aged 55-64 years. Severe clinical events in p.N215S patients were mainly cardiac (males 31%, females 8%) while renal and cerebrovascular events were rare. Renal impairment occurred in 17% of p.N215S males (mostly in patients aged 65-74 years), and rarely in females (3%).Conclusionp.N215S is a disease-causing mutation with severe clinical manifestations found primarily in the heart. Cardiac involvement may become as severe as in classic Fabry patients, especially in males

Fluctuations in phenylalanine concentrations in phenylketonuria: a review of possible relationships with outcomes.

International audience; Fluctuations in blood phenylalanine concentrations may be an important determinant of intellectual outcome in patients with early and continuously treated phenylketonuria (PKU). This review evaluates the studies on phenylalanine fluctuations, factors affecting fluctuations, and if stabilizing phenylalanine concentrations affects outcomes, particularly neurocognitive outcome. Electronic literature searches of Embase and PubMed were performed for English-language publications, and the bibliographies of identified publications were also searched. In patients with PKU, phenylalanine concentrations are highest in the morning. Factors that can affect phenylalanine fluctuations include age, diet, timing and dosing of protein substitute and energy intake, dietary adherence, phenylalanine hydroxylase genotype, changes in dietary phenylalanine intake and protein metabolism, illness, and growth rate. Even distribution of phenylalanine-free protein substitute intake throughout 24h may reduce blood phenylalanine fluctuations. Patients responsive to and treated with 6R-tetrahydrobiopterin seem to have less fluctuation in their blood phenylalanine concentrations than controls. An increase in blood phenylalanine concentration may result in increased brain and cerebrospinal fluid phenylalanine concentrations within hours. Although some evidence suggests that stabilization of blood phenylalanine concentrations may have benefits in patients with PKU, more studies are needed to distinguish the effects of blood phenylalanine fluctuations from those of poor metabolic control

Mutations of the Mitochondrial-tRNA Modifier MTO1 Cause Hypertrophic Cardiomyopathy and Lactic Acidosis

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

Fabry disease and COVID-19: International expert recommendations for management based on real-world experience

The rapid spread of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 has raised questions about Fabry disease (FD) as an independent risk factor for severe COVID-19 symptoms. Available real-world data on 22 patients from an international group of healthcare providers reveals that most patients with FD experience mild-to-moderate COVID-19 symptoms with an additional complication of Fabry pain crises and transient worsening of kidney function in some cases; however, two patients over the age of 55 years with renal or cardiac disease experienced critical COVID-19 complications. These outcomes support the theory that pre-existent tissue injury and inflammation may predispose patients with more advanced FD to a more severe course of COVID-19, while less advanced FD patients do not appear to be more susceptible than the general population. Given these observed risk factors, it is best to reinforce all recommended safety precautions for individuals with advanced FD. Diagnosis of FD should not preclude providing full therapeutic and organ support as needed for patients with FD and severe or critical COVID-19, although a FD-specific safety profile review should always be conducted prior to initiating COVID-19-specific therapies. Continued specific FD therapy with enzyme replacement therapy, chaperone therapy, dialysis, renin–angiotensin blockers or participation to clinical trials during the pandemic is recommended as FD progression will only increase susceptibility to infection. In order to compile outcome data and inform best practices, an international registry for patients affected by Fabry and infected by COVID-19 should be established

Experience of the NPC Brazil network with a comprehensive program for the screening and diagnosis of Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is a lysosomal disorder caused by impaired cholesterol metabolism. Levels of lysosphingomyelin 509 (LysoSM509) have been shown elevated in dried blood spots (DBS) of NPC and acid sphingomyelinase deficiency patients. In this study, we report our experience using a two-tier approach (1st tier is the quantification of lysoSM509 by ultra-performance liquid chromatography tandem mass spectrometry followed by the 2nd tier with next-generation sequencing of the NPC1 and NPC2 genes). DBS samples from 450 suspected patients were received by the NPC Brazil network. Of these, 33 samples had elevated levels of lysoSM509, and in 25 of them, variants classified as pathogenic, likely pathogenic, or of unknown significance were identified in the NPC1 or NPC2 genes by next-generation sequencing. The quantification of lysoSM509 in DBS as a first-tier test for the diagnosis of NPC followed by molecular analysis of the NPC1 and NPC2 genes almost doubled the detection rate when compared to the performance of chitotriosidase activity as a first-tier biomarker, and it could likely be increased with the addition of a third tier with MLPA of the two genes involved. This strategy seems suitable for the neonatal screening (NBS) of NPC if this disease is eventually adopted by NBS programs