Impact of newborn screening for fatty acid oxidation disorders on neurological outcome: A Belgian retrospective and multicentric study.

Fatty acid oxidation (FAO) disorders are autosomal recessive genetic disorders affecting either the transport or the oxidation of fatty acids. Acute symptoms arise during prolonged fasting, intercurrent infections, or intense physical activity. Metabolic crises are characterized by alteration of consciousness, hypoglycemic coma, hepatomegaly, cardiomegaly, arrhythmias, rhabdomyolysis, and can lead to death. In this retrospective and multicentric study, the data of 54 patients with FAO disorders were collected. Overall, 35 patients (64.8%) were diagnosed after newborn screening (NBS), 17 patients on clinical presentation (31.5%), and two patients after family screening (3.7%). Deficiencies identified included medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (75.9%), very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (11.1%), long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency (3.7%), mitochondrial trifunctional protein (MTP) deficiency (1.8%), and carnitine palmitoyltransferase 2 (CPT 2) deficiency (7.4%). The NBS results of 25 patients were reviewed and the neurological outcome of this population was compared with that of the patients who were diagnosed on clinical presentation. This article sought to provide a comprehensive overview of how NBS implementation in Southern Belgium has dramatically improved the neurological outcome of patients with FAO disorders by preventing metabolic crises and death. Further investigations are needed to better understand the physiopathology of long-term complications in order to improve the quality of life of patients and to ensure optimal management.

Blocking of frizzled signaling with a homologous peptide fragment of wnt3a/wnt5a reduces infarct expansion and prevents the development of heart failure after myocardial infarction.

BACKGROUND: The molecular pathways that control the wound healing after myocardial infarction (MI) are not completely elucidated. One of these pathways is the Wnt/Frizzled pathway. In this study, we evaluated Frizzled as a novel therapeutic target for MI. These Frizzled proteins act as receptors for Wnt proteins and were previously shown to be expressed in the healing infarct. METHODS AND RESULTS: Wnt/Frizzled signaling has been studied for decades, but synthetic ligands that interfere with the interaction between Wnts and Frizzled have not been described to date. Here we report the selection of 3 peptides derived from regions of high homology between Wnt3a and Wnt5a that act as antagonists for Frizzled proteins. UM206, the peptide with the highest affinity, antagonized the effect of Wnt3a and Wnt5a in different in vitro assays. Administration of UM206 to mice for 5 weeks, starting immediately after the induction of MI, reduced infarct expansion and increased the numbers of capillaries and myofibroblasts in the infarct area. Moreover, heart failure development was inhibited by this therapy. CONCLUSIONS: Blocking of Frizzled signaling reduces infarct expansion and preserves cardiac function after MI. Our findings underscore the potential of Frizzled receptors as a target for pharmacotherapy of cardiac remodeling after MI.

Belgian rare diseases plan in clinical pathology: identification of key biochemical diagnostic tests and establishment of reference laboratories and financing conditions.

BACKGROUND: One objective of the Belgian Rare Diseases plan is to improve patients' management using phenotypic tests and, more specifically, the access to those tests by identifying the biochemical analyses used for rare diseases, developing new financing conditions and establishing reference laboratories. METHODS: A feasibility study was performed from May 2015 until August 2016 in order to select the financeable biochemical analyses, and, among them, those that should be performed by reference laboratories. This selection was based on an inventory of analyses used for rare diseases and a survey addressed to the Belgian laboratories of clinical pathology (investigating the annual analytical costs, volumes, turnaround times and the tests unavailable in Belgium and outsourced abroad). A proposal of financeable analyses, financing modalities, reference laboratories' scope and budget estimation was developed and submitted to the Belgian healthcare authorities. After its approval in December 2016, the implementation phase took place from January 2017 until December 2019. RESULTS: In 2019, new reimbursement conditions have been published for 46 analyses and eighteen reference laboratories have been recognized. Collaborations have also been developed with 5 foreign laboratories in order to organize the outsourcing and financing of 9 analyses unavailable in Belgium. CONCLUSIONS: In the context of clinical pathology and rare diseases, this initiative enabled to identify unreimbursed analyses and to meet the most crucial financial needs. It also contributed to improve patients' management by establishing Belgian reference laboratories and foreign referral laboratories for highly-specific analyses and a permanent surveillance, quality and financing framework for those tests.

Inter-laboratory analytical improvement of succinylacetone and nitisinone quantification from dried blood spot samples.

BACKGROUND: Nitisinone is used to treat hereditary tyrosinemia type 1 (HT-1) by preventing accumulation of toxic metabolites, including succinylacetone (SA). Accurate quantification of SA during newborn screening is essential, as is quantification of both SA and nitisinone for disease monitoring and optimization of treatment. Analysis of dried blood spots (DBS) rather than plasma samples is a convenient method, but interlaboratory differences and comparability of DBS to serum/plasma may be issues to consider. METHODS: Eight laboratories with experience in newborn screening and/or monitoring of patients with HT-1 across Europe participated in this study to assess variability and improve SA and nitisinone concentration measurements from DBS by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantification of nitisinone from both DBS and plasma was performed to assess sample comparability. In addition, efforts to harmonize laboratory procedures of SA and nitisinone quantifications during 5 rounds of analysis are described. RESULTS: Nitisinone levels measured from DBS and plasma strongly correlated (R 2 = 0.93). Due to partitioning of nitisinone to the plasma, levels were higher in plasma by a factor of 2.34. In the initial assessment of laboratory performance, all had linear calibrations of SA and nitisinone although there was large inter-laboratory variability in actual concentration measurements. Subsequent analytical rounds demonstrated markedly improved spread and precision over previous rounds, an outcome confirmed in a final re-test round. CONCLUSION: The study provides guidance for the determination of nitisinone and SA from DBS and the interpretation of results in the clinic. Inter-laboratory analytical harmonization was demonstrated through calibration improvements.

Parenteral hydroxocobalamin dose intensification in five patients with different types of early onset intracellular cobalamin defects: Clinical and biochemical responses.

Intracellular cobalamin metabolism (ICM) defects can be present as autosomal recessive or X-linked disorders. Parenteral hydroxocobalamin (P-OHCbl) is the mainstay of therapy, but the optimal dose has not been determined. Despite early treatment, long-term complications may develop. We have analyzed the biochemical and clinical responses in five patients with early onset of different types of ICM defects (cblC: patients 1-3; cblA: patient 4; cblX: patient 5) following daily P-OHCbl dose intensification (DI). In patient 4, P-OHCbl was started at age 10 years and in patient 5 at age 5 years. OHCbl was formulated at either, 5, 25, or 50 mg/mL. P-OHCbl was intravenously or subcutaneously (SQ) delivered, subsequently by placement of a SQ injection port except in patient 4. In all patients, homocysteine and methylmalonic acid levels, demonstrated an excellent response to various P-OHCbl doses. After age 36 months, patients 1-3 had a close to normal neurological examination with lower range developmental quotient. In patient 3, moderate visual impairment was present. Patient 4, at age 10 years, had normal renal, visual and cognitive function. In cblX patient 5, epilepsy was better controlled. In conclusion, P-OHCbl-DI caused an excellent control of metabolites in all patients. In the three cblC patients, comparison with patients, usually harboring identical genotype and similar metabolic profile, was suggestive of a positive effect, in favor of clinical efficacy. With P-OHCbl-DI, CblA patient has been placed into a lower risk to develop renal and optic impairment. In cblX patient, lower P-OHCbl doses were administrated to improve tolerability.

Clinical implementation of gene panel testing for lysosomal storage diseases.

BACKGROUND: The diagnostic workup in patients with a clinical suspicion of lysosomal storage diseases (LSD) is often difficult due to the variability in the clinical phenotype. The gold standard for diagnosis of LSDs consists of enzymatic testing. However, due to the sequential nature of this methodology and inconsistent genotype-phenotype correlations of certain LSDs, finding a diagnosis can be challenging. METHOD: We developed and clinically implemented a gene panel covering 50 genes known to cause LSDs when mutated. Over a period of 18 months, we analyzed 150 patients who were referred for LSD testing and compared these results with the data of patients who were previously enrolled in a scheme of classical biochemical testing. RESULTS: Our panel was able to determine the molecular cause of the disease in 22 cases (15%), representing an increase in diagnostic yield compared to biochemical tests developed for 21 LSDs (4.6%). We were furthermore able to redirect the diagnosis of a mucolipidosis patient who was initially suspected to be affected with galactosialidosis. Several patients were identified as being affected with neuronal ceroid lipofuscinosis, which cannot readily be detected by enzyme testing. Finally, several carriers of pathogenic mutations in LSD genes related to the disease phenotype were identified as well, thus potentially increasing the diagnostic yield of the panel as heterozygous deletions cannot be detected. CONCLUSION: We show that the implementation of a gene panel for LSD diagnostics results in an increased yield in comparison to classical biochemical testing. As the panel is able to cover a wider range of diseases, we propose to implement this methodology as a first-tier test in cases of an aspecific LSD presentation, while enzymatic testing remains the first choice in patients with a more distinctive clinical presentation. Positive panel results should however still be enzymatically confirmed whenever possible.

Angiotensin AT4 receptor ligand interaction with cystinyl aminopeptidase and aminopeptidase N: [125I]Angiotensin IV only binds to the cystinyl aminopeptidase apo-enzyme.

Due to its high affinity for [(125)I]Angiotensin IV, cystinyl aminopeptidase (CAP) has recently been assigned as the 'angiotensin AT(4) receptor'. Since the aminopeptidase N (AP-N) activity is also susceptible to inhibition by Angiotensin IV, it might represent an additional target for this peptide. Based on [(125)I]Angiotensin IV binding and catalytic activity measurements, we compared the ligand interaction properties of recombinant human CAP and human AP-N. Both enzymes displayed distinct pharmacological profiles. Although their activity is inhibited by Angiotensin IV and LVV-hemorphin 7, both peptides are more potent CAP-inhibitors. On the other hand, substance P and l-methionine have a higher potency for AP-N. High affinity binding of [(125)I]Angiotensin IV to CAP occurs in the presence of chelators but not to AP-N in either the absence or presence of chelators. These differences were exploited to determine whether CAP and/or AP-N are present in different cell lines (CHO-K1, COS-7, HEK293, SK-N-MC and MDBK). We provide evidence that CAP predominates in these cell lines and that, comparatively, CHO-K1 cells display the highest level of this enzyme.

Metal ion modulation of cystinyl aminopeptidase.

Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2'-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32+/-1.78 and 2.10+/-0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N',N'',N''-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as 'AT4 receptor binding', only occurs for the cystinyl aminopeptidase apoenzyme.

Synergistic modulation of cystinyl aminopeptidase by divalent cation chelators.

Membranes of Chinese hamster ovary (CHO-K1) cells were used to study the opposite modulation of enzyme activity and [125I]Ang IV binding to cystinyl aminopeptidase (EC 3.4.11.3) by divalent cation chelators. Whereas ethylene diamine tetraacetic acid (EDTA) or ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) alone only slightly affected the enzyme activity, 1,10-phenanthrolin (1,10-PHE) produced a complete and concentration-dependent inhibition. Interestingly EDTA (> or =0.05 mM) or EGTA (> or =0.15 mM) enhanced the inhibitory effect of 1,10-PHE. Two-site analysis of the corresponding inhibition curves revealed that EDTA and EGTA converted enzymes with low sensitivity towards 1,10-PHE into enzymes with high sensitivity. The combined inhibition by EDTA (0.1 mM) and 1,10-PHE (0.1 mM) could be prevented and reversed by addition of Zn2+ (at about 0.04-0.1 mM). In contrast, specific binding of [125I]Ang IV was enhanced in the presence of 1,10-PHE. Binding was only slightly affected by EDTA or EGTA alone. Furthermore, the stimulatory effect of 1,10-PHE was potentiated by EDTA (> or =0.05 mM) as well as EGTA (> or =0.15 mM). In the presence of EDTA (0.1 mM) and 1,10-PHE (0.1 mM), specific [125I]Ang IV binding was completely inhibited by Zn2+ (IC50= 39.7 +/- 6.2 microM). The present data show that divalent cations such as Zn2+ are essential for the enzyme activity of cystinyl aminopeptidase and inhibitory for [125I]Ang IV binding. Modulation of the effects of 1,10-PHE by other chelators such as EDTA or EGTA, suggests that, in addition to the binding site for zinc in the catalytic site, cystinyl aminopeptidase also bears a regulatory divalent cation binding site.

Endogenous cystinyl aminopeptidase in Chinese hamster ovary cells: characterization by [125I]Ang IV binding and catalytic activity.

The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC 3.4.11.3), a membrane-associated zinc-dependent metallopeptidase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT4 receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [125I]Ang IV and by measuring the cleavage of L-leucine-p-nitroanilide. The equilibrium dissociation constant of [125I]Ang IV in saturation binding studies (KD= 0.90 nM) was similar to the value (KD= 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the "AT4 receptor" ligands (Ang IV > divalinal1-Ang IV approximately LVV-hemorphin-7 approximately LVV-hemorphin-6 > Ang (3-7) > Ang III > Ang (4-8)) but not by AT1/AT2 receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang IV and other "AT4 receptor" ligands (Ang IV > Ang III approximately divalinal1-Ang IV approximately Ang (3-7) approximately LVV-hemorphin-7 > Ang (4-8) approximately LVV-hemorphin-6). High affinity binding of [125I]Ang IV required the presence of metal chelators and the ligands such as Ang IV and LVV-hemorphin-7 displayed higher potency in the binding studies as in the enzyme assay. This difference in potency varied from one peptide to another. These pharmacological properties match those previously reported for the recombinantly-expressed human cystinyl aminopeptidase in embryonal kidney cells.

Wnt/frizzled signalling modulates the migration and differentiation of immortalized cardiac fibroblasts.

AIMS: The Wnt/frizzled (Fzd) signal transduction cascade has been implicated in the proliferation, differentiation, and migration of many cell types, but the role of this pathway in cardiac fibroblast differentiation is not known. Our lab previously showed an up-regulation of Fzd-1 and -2 expression in myofibroblasts after myocardial infarction (MI), indicating a potential role for the Fzd receptor in fibroblast-myofibroblast differentiation. The present study was performed to further define the role of specific Wnt and Fzd proteins in the proliferation, migration, and differentiation of cardiac fibroblasts. METHODS AND RESULTS: Because primary fibroblasts become senescent after a few passages and are difficult to transfect, we immortalized rat cardiac fibroblasts with telomerase [cardiac fibroblasts immortalized with telomerase (CFIT)]. Proliferation of CFIT was not significantly influenced by Wnt/Fzd signalling. The migration, however, was attenuated by all Wnt/Fzd combinations tested. Also, specific Wnt/Fzd combinations modulated the expression of the following myofibroblast markers: collagen Ialpha1, collagen III, fibronectin and its splice variants, and alpha-smooth muscle actin. CONCLUSION: The results indicate that myofibroblast migration and differentiation, but not proliferation, can be modulated by interventions in Wnt/Fzd signalling. Therefore, Wnt/Fzd signalling may serve as a novel therapeutic target to ameliorate wound healing after MI.